Cardiac norepinephrine and intrinsic properties of isolated hypertrophied hearts from DOCA hypertensive rats

Alterations in cardiac norepinephrine (NE) levels often accompany cardiac hypertrophy. The present study addresses the question of whether such differences in endogenous NE might be responsible for hypertrophy-dependent differences in acutely measured mechanical and metabolic properties of isolated rat heart preparations. Hypertrophied hearts (H) of deoxycorticosterone acetate-hypertensive rats and normal hearts (N) of sham-treated rats were perfused in Langendorff fashion with atrial tissue removed. Assessment of ventricular NE after 3 to 5 min indicated that the concentration was lower in H than N but total content of the left ventricle was not significantly different. Assessment of ventricular NE after three hours suggested that some washout or depletion of NE occurred in H but not N. Mechanical and metabolic characteristics of the isovolumic preparation were determined. When compared to N, H had greater spontaneous ventricular beating rates and, when paced with field stimuli, higher systolic pressures, longer relaxation times and mechanical refractory periods, and greater oxygen consumption. When the latter was normalized for ventricular mass and systolic pressure, H utilized less oxygen/g/100 mmHg pressure than N. Elimination of the acute effects of endogenous NE by either blockade (propranolol) or depletion (reserpine) eliminated the difference in ventricular automaticity between H and N, but did not influence the other differences (or similarities) between H and N. We conclude that aside from changes in ventricular automaticity, hypertrophy-dependent alterations in cardiac mechanical and metabolic properties are not likely to be a result of differing acute influences of endogenous norepinephrine.     

Correlation of contractile dysfunction and abnormal tissue energy metabolism during hypoperfusion with norepinephrine in isolated rat hearts: differences between normal and diabetic hearts.

The relationship of myocardial high-energy phosphate depletion and lactate accumulation with contractile dysfunctions was investigated in streptozotocin-diabetic (DM) and normal rat hearts. The isolated hearts were perfused with 10(-6) M norepinephrine (NE) at various low-flow rates (0.4-6 ml/min/g heart wt) for 1 h. Left ventricular pressure (LVP) and contractile force (CF) were monitored, through a water-filled balloon in LV and through a hook attached to the apex, respectively. In DM hearts resting CF (diastolic tension) increased, when the perfusion flow rate was reduced below 6 ml and reached a maximum at a flow rate of less than 3 ml. The large increase in LV stiffness correlated with an elevation in diastolic LVP. In normal hearts these parameters were elevated at a flow rate below 1 ml. A flow-dependent decrease in developed CF was more prominent in DM than in normal hearts, while developed LVP and perfusion pressure were slightly higher in DM hearts with a marked increase in the LV stiffness. A flow-dependent decrease in high-energy phosphates and increases in inorganic phosphate and lactate were more prominent in the inner than in the outer layer of LV free wall in both groups. The change of ATP in the inner layer was greater, while increases of lactate in both layers were smaller in DM hearts. Changes in mechanical parameters correlated well with the ATP decrease and lactate increase in the inner layer in both groups. The correlation curves, however, were not coincidental: at the same low ATP and high lactate level, the LV stiffness was higher in DM hearts. Results indicate that DM hearts are more susceptible to flow-reduction with NE and depletion of total ATP in their tissue, and easily suffer from increased LV stiffness. This cannot be explained by the rate of decrease in total ATP and lactate accumulation alone.     

Influence of Bretschneider's cardioplegia on norepinephrine release from isolated perfused guinea-pig hearts

It was the aim of the present study to investigate the influence of Bretschneider's cardioplegia on norepinephrine (NE) release [determined by high pressure liquid chromatography (HPLC) and electrochemical detection] in isolated perfused guinea-pig hearts. The following resulted were noted. (1) Calcium-dependent exocytotic NE release evoked by electrical field stimulation (12 Hz, 1 min) was completely suppressed after only 3 min of normothermic (37.5 degrees C) Bretschneider's cardioplegia. (2) Stop-flow ischemia is associated with a substantial calcium-independent, non-exocytotic NE release, which is regarded as a sodium-dependent carrier-mediated process. Accordingly, it is inhibited by blockers of the sodium/proton-exchanger (e.g. amiloride) and the neuronal uptake1-carrier (e.g. desipramine). Compared with stop-flow ischemia alone, cardioplegia with 3 min of Bretschneider's histidine-tryptophan-ketoglutarate (HTK)-solution preceding stop-flow enhanced NE release at all stop-flow durations (10-90 min) investigated (e.g. after 30 min of normothermic Bretschneider's cardioplegia: 1070+/-41 pmol/g, n = 45, v stop-flow alone: 764+/-48 pmol/g, n = 27, P<0.05). The NE concentrations determined in the cardiac effluent upon reperfusion followed a typical first order kinetic indicating that the transmitter release had already occurred during stop-flow. Hypothermia reduced NE release in a temperature-dependent manner down to intramyocardial temperatures of 2 7.5 degrees C. NE release evoked by Bretschneider's cardioplegia still exceeded that induced by stop-flow ischemia alone by up to 60%. The NE release evoked by Bretschneider's cardioplegia and stop-flow ischemia was calcium-independent. However, it was significantly reduced by desipramine and amiloride, but both agents had a more pronounced inhibitory effect on NE release evoked by stop-flow ischemia alone. (3) This difference may be due to an intrinsic effect of Bretschneider's HTK-solution, as continuous administration of normothermic Bretschneider's HTK-solution induced a substantial NE release which was neither calcium-dependent nor inhibited by blockade of either uptake1 or sodium/proton-exchange. It is concluded that Bretschneider's cardioplegia is not neuroprotective, as it even augments the stop-flow ischemia-induced nonexocytotic NE release.     

Effect of physical training on norepinephrine turnover in tissues of normal and diabetic rats

This study was designed to examine the influence of physical training on the norepinephrine turnover rate in heart, pancreas, liver, and gastrocnemius muscles of normal and diabetic male rats at rest. Diabetes was induced with the IV injection of streptozotocin (45 mg/kg) and physical training was done on a treadmill according to a ten-week program. Norepinephrine turnover rate of tissues was estimated by following over time the decay in the specific activity of norepinephrine after a single IV bolus of tritiated norepinephrine (30 microCi/kg). Plasma glucose, insulin, and glucagon levels were also measured at the time of death. Although training caused a reduction in the plasma glucose values of diabetic rats, no changes in norepinephrine turnover rate were observed after the conditioning program. On the other hand, diabetes was associated with a significant 30% to 40% decrease in the pancreatic norepinephrine turnover rate. It is concluded that the beneficial effects of physical training on diabetes mellitus cannot be explained by adaptive changes in the sympathetic nervous system activity and that further work will be necessary to elucidate the mechanism whereby streptozotocin diabetes diminishes the pancreatic norepinephrine turnover.     

Metabolism of palmitate in isolated working hearts from spontaneously diabetic "BB" Wistar rats

Myocardial fatty acid metabolism was studied in spontaneously-diabetic "BB" Wistar rats. The study involved 4 groups: control Wistar rats, nondiabetic littermates of "BB" Wistar rats, insulin-treated diabetic "BB" rats, and diabetic "BB" rats in which insulin treatment was removed 24 hours prior to study (uncontrolled diabetes). Hearts were perfused for 30 minutes as isolated working hearts in perfusate containing 1.2 mM (1-14C)-palmitate bound to 3% albumin, and 11 mM glucose. Palmitate oxidative rates, calculated as micromoles palmitate oxidized per gram dry weight per minute, were significantly decreased in both diabetic groups (0.447 +/- 0.043 and 0.528 +/- 0.038 in uncontrolled diabetic and treated diabetic versus 0.584 +/- 0.032 and 0.629 +/- 0.033 in nondiabetic littermate and control rats, respectively). This decrease was accompanied, however, by a significant decrease in the heart rate of these 2 groups when compared with control or nondiabetic animals. If the decreased heart function in the diabetic animals was accounted for, no decrease in palmitate oxidative rates occurred, suggesting that fatty acid oxidative metabolism is not impaired in the diabetic myocardium. In the uncontrolled diabetic rats, an increased rate of palmitate incorporation into myocardial triglycerides was seen compared with treated diabetic, nondiabetic littermates, and control rats (8.5 +/- 0.3 mumol/g dry wt/30 min versus 4.8 +/- 0.3, 5.9 +/- 0.7, and 5.7 +/- 0.3, respectively). Myocardial levels of coenzyme A were elevated in the uncontrolled diabetic rats compared with all other groups (647 +/- 25 nmol/g dry wt versus 484 +/- 27, 508 +/- 56, and 534 +/- 9, in treated diabetic, nondiabetic, and control rats, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of chronic dobutamine administration on hearts of normal and hypertensive rats

We have previously shown that physical conditioning in the rat improves cardiac mechanics and biochemistry and normalizes the cardiac contractile protein abnormalities associated with renovascular hypertension. Since chronic adrenergic stimulation with dobutamine simulates some aspects of physical conditioning, this study was undertaken to investigate the effects of chronic dobutamine administration on normal and hypertensive rat hearts. Four groups of female animals were studied: controls, dobutamine-treated (2 mg/kg twice daily), renovascular hypertensives, and dobutamine-treated hypertensives. Animals were killed after 8-10 weeks and cardiac histology, myosin biochemistry, and mechanics in an isolated heart perfusion apparatus were studied. Dobutamine, unlike hypertension, was not associated with histological evidence of myocardial damage but did increase cardiac mass by 10% and calcium-activated myosin ATPase activity by 13%. Hypertension was associated with a 24% increase in mass, a 24% decrease in ATPase activity, and a shift in the myosin isoenzyme pattern from V1 to V3. The combined stimuli caused additive hypertrophy (44%) and normalized myosin biochemistry and isomyosin distribution. Dobutamine treatment was not associated with significant improvements in pump or muscle function in control or hypertensive hearts. Thus chronic dobutamine treatment, like physical conditioning, induces a physiological cardiac hypertrophy in rats that is associated with improved myosin enzymology and normalization of the contractile protein abnormalities associated with hypertension. Unlike physical conditioning, however, these biochemical alterations do not result in improved contractile function as measured in an isolated buffer-perfused heart apparatus.     

Depressed function of isolated hearts from hyperdynamic septic rats

The aim of this study was to determine if hearts removed from rats in sustained hypermetabolic sepsis possessed normal physiological reserves as defined by Frank-Starling curves. The resting cardiac output of these hearts is elevated in vivo and this is mediated by an increased heart rate. In this study mechanical function was assessed under in vitro conditions in which preload could be varied and controlled. Isolated perfused working hearts from septic animals showed depressed cardiac performance, both in terms of peak systolic pressure development and cardiac output, over a range of left atrial filling pressures. In spite of this depressed function, most hearts from septic animals were able to recover cardiac output and pressure development following an acute ischemic episode. Thus, a loss of myocardial reserve is demonstrable in hearts isolated from septic rats, even though mean arterial blood pressure, cardiac output, and coronary blood flow are well maintained in vivo. This dysfunction is primarily evidenced by an inability to respond appropriately to increased preloads.     

Excitability of isolated hearts from rats during postnatal development

INTRODUCTION: After birth, cardiac myocytes undergo substantial growth and differentiation that affect their stimulation threshold. Cells from younger animals have a higher stimulation threshold than cells from adults. The aim of this work was to compare the excitability of isolated hearts of rats during postnatal development by measuring the stimulation threshold at several pulse durations. METHODS AND RESULTS: Stimulation threshold of isolated hearts were measured at eight different pulse durations between 0.1 to 20 msec. For each heart, a strength-duration curve was constructed and data were fitted using both Weiss-Lapicque and Blair models. Analysis of variance showed significant age-dependent differences in both rheobase field (E(reob)) and chronaxie (c). E(reob) decreased while c increased during development (E(reob) was 0.21, 0.16, 0.13, 0.10, and 0.09 V/cm and c was 2.0, 2.2, 2.3, 2.7, and 3.2 msec for rats aged 1, 2, 4, 8, and 20 weeks, respectively). There was a decrease in the threshold field with heart weight between 0.1 and 0.7 g, whereas the threshold was almost constant in the range from 0.7 to 2.0 g. Estimation of the energy density needed to defibrillate the heart was performed and appeared to be higher for younger than for adult animals. CONCLUSION: Hearts from younger animals have higher stimulation threshold than those from adults. This probably is due to changes in the cellular threshold as a result of maturation. The smaller excitability of younger hearts can have consequences with regard to the energy levels required for younger patients.     

Increased prostacyclin release from perfused hearts of acutely diabetic rats

Summary The release of prostacyclin and PGE₂ from the isolated perfused hearts of acutely diabetic (streptozotocin 100 mg/kg) rats was studied and compared with hearts from control animals. Prostacyclin and PGE₂ were measured by a differential bioassay technique. No basal release of either prostaglandin was detected. However, after addition of arachidonic acid, a dose dependent release of prostacyclin and PGE₂ was noted. Prostacyclin was identified as the major prostaglandin. Release of prostacyclin and PGE₂ from acutely diabetic rat hearts was increased 2–3 times compared to control hearts. No release of prostaglandin endoperoxides was observed in either group of hearts.     

Effects of synthetic rat C-peptide in normal and diabetic rats

Summary The effects of synthetic rat C-peptide 1 and C-peptide 2 on plasma insulin and blood glucose concentrations in the rat were studied. Infusion of rat C-peptide (500g·h^-1· kg^-1) diminished glucose induced increase of plasma insulin by 56% (15.2±0.9 versus 6.6± 0.6 ng/ml, p<0.01, mean±SEM). Somatostatin infused at a rate of 50 g·h^-1·kg^-1 body weight inhibited glucose-induced insulin secretion by 33%. In the presence of a mixture of both C-peptides or somatostatin, blood glucose after intravenous glucose was higher than in the control experiments. In alloxan-diabetic rats, C-peptide (160 g/kg) significantly increased and prolonged the hypoglycaemic effect of exogenous insulin. It is suggested that C-peptide may not be a biologically inert substance.     

Effects of cortisol and progesterone on insulin binding and lipogenesis in adipocytes from normal and diabetic rats

Cortisol implants in normal and diabetic rats reduced body weight, adiposity, insulin receptor concentration and both basal and insulin-stimulated rates of lipogenesis in isolated adipocytes, whilst insulin sensitivity was unchanged. In normal but not diabetic rats these changes were accompanied by increased serum glucose and insulin concentrations. In contrast, progesterone implants in normal and diabetic rats increased body weight gain, adiposity, insulin receptor concentration and both basal and insulin-stimulated rates of lipogenesis in adipose tissue, again without affecting insulin sensitivity. Progesterone did not affect serum insulin concentrations in normal or diabetic rats but accelerated the decline in serum glucose concentrations which occurred during an overnight fast in diabetic rats. The results suggest that cortisol inhibits lipogenesis in adipose tissue without affecting insulin sensitivity, cortisol reduces insulin binding in adipose tissue without a requirement for hyperinsulinaemia, which might itself indirectly lead to down-regulation of the insulin receptor, and in diabetic rats progesterone stimulates lipogenesis in adipose tissue without any increase in food intake or serum insulin concentrations suggesting that progesterone may have a direct anabolic role in adipose tissue.     

Energy dependent insulin binding, internalization and degradation in isolated cardiac myocytes from normal and diabetic rats

Insulin binding to isolated cardiac myocytes from normal and streptozotocin-induced diabetic rats was investigated. We found that at high affinity sites, the maximum numbers of insulin binding sites per cell are 33 000 and 22 000 for normal and diabetic myocytes, respectively with no discernible difference in receptor affinity. However, since the yield of myocytes from the diabetic heart was only 1/3 of the normal heart, it is suspected that the insulin function in the diabetic heart may be significantly lower than that in the normal heart. Chloroquine was found to markedly decrease insulin degradation with concomitant increase in net insulin uptake by isolated myocytes. This suggests that insulin degradation may take place within lysosomes after insulin is internalized. To determine whether internalization of insulin in myocytes is an energy dependent process, insulin binding and subsequent degradation were assessed in cells depleted of ATP by treatment with various metabolic inhibitors (2,4-dinitrophenol, NaF and iodoacetic acid). Depletion of the cellular ATP level resulted in a decrease in both insulin uptake and degradation. In diabetic myocytes, the general relationship between cellular ATP level and insulin uptake and degradation was similar to that found in normal myocytes. However, in diabetic myocytes, the cellular ATP level and insulin uptake were lower, but insulin degradation was greater than in normal myocytes. Insulin uptake by normal and ATP depleted cells at 4 degrees C (16 h) was lower than at 37 degrees C (1 h), while the ATP level was almost the same at both temperatures. This suggests that the internalization of insulin is a temperature as well as an ATP dependent process.     

Investigations on glucose uptake in isolated human leucocytes from normal and diabetic subjects

Summary 1. Leucocyte preparations have been obtained from the blood of 135 healthy control persons and 10 poorly-controlled insulin-dependent diabetics according to a procedure described earlier. Cells were suspended in a Krebs-Ringer-Bicarbonate-Buffer. Glucose uptake, lactate production and cell glucose space were determined after incubating the cells for 1 h at 37° C in an atmosphere of O₂/CO₂ (95/5). Extracellular water-space of the cell sediment was corrected by measuring U-¹⁴C-sucrose levels in the medium before and after addition to the cell sediment. — 2. The cells showed intact structures and amoeboid motility under the light-microscope as well as under the phase-contrast-microscope. During an incubation lasting 1 h, the ATP/ADP quotient diminished by about 20%; the following metabolites: glucose-6-phosphate, fructose-1,6-diphosphate, 1,3-phosphoglycerate, pyruvate and lactate increased slightly up to markedly, especially glucose-6-phosphate and lactate. — 3. Sources of methodological errors were investigated in preliminary experiments. Disregarding corrections for the extracellular space of the cell sediments led to a dilution effect which imitates an apparent glucose uptake. For measurements of intracellular glucose and cell glucose space the procedure of Crofford and Renold provided the best results. Falsely high values for cell glucose have been found using the glucose oxidase reaction. — 4. Glucose uptake of healthy leucocytes increases at first steeply, later on less with rising medium glucose concentrations. Addition of insulin (50–500 mU/ml) gave no consistent effects. At medium glucose concentrations of 400 and 600 mg/100 ml intracellular glucose could be demonstrated with statistical significance. Intracellular glucose and cell glucose space were not affected by insulin to any marked degree. Determinations of lactate production indicated that human leucocytes utilize the largest portion of glucose via glycolysis. — 5. At medium glucose concentrations of 600 mg/100 ml leucocytes from diabetics who had received no insulin for 14 h prior to incubation showed a significant diminution of glucose uptake. — 6. Theoretical curves for inward transport and phosphorylation have been calculated from the data for glucose uptake and intracellular glucose concentration.The authors are greatly indepted to Prof. Wilbrandt, Department of Pharmacology, University Bern, for advice and discussion.     

Calcium uptake by sarcoplasmic reticulum isolated from hearts of septic rats

Myocardial sarcoplasmic reticulum (SR) plays a critical role in the regulation of the cytosolic calcium fluctuations that occur during the cardiac cycle. One function of the SR is to lower the calcium concentration so that myocardial relaxation and thus ventricular filling can occur. The aim of the present study was to determine if hyperdynamic sepsis induced a decrease in the capacity of SR to take up calcium. This defect would result in decreased ventricular filling and thus decreased cardiac output, as has previously been shown in isolated perfused working hearts removed from septic rats. Therefore, rats were anesthetized with ether, and sepsis was induced by the injection of an aliquot of a fecal homogenate into the peritoneal cavity. Control animals either underwent surgery and received an aliquot of sterilized fecal inoculum (sham) or were untreated (no surgery). On day 2 after surgery, animals were anesthetized with pentobarbital, and hearts were removed, weighted, and SR isolated. The rate of uptake of 45Ca2+ by SR from septic rats was not depressed compared to controls but in fact was elevated. Maximum 45Ca2+ accumulated by the SR and Ca2+-stimulated ATPase activity were similar in SR from control and septic hearts. These results suggest that the contractile dysfunction noted in the myocardium in early sepsis is probably not due to inadequate SR removal of Ca2+ during diastole.     

Chronic effects of tolbutamide on myocardial tension during ischemia and reperfusion in perfused hearts isolated from insulin dependent and non insulin dependent diabetic rats

The chronic effects of tolbutamide on myocardial contractility of the diabetic heart during ischemia and reperfusion were evaluated in perfused, isolated rat hearts. Five experimental groups were used: (1) control rats (C), (2) insulin dependent diabetic rats (IDDM, single intravenous injection of 60 mg/kg streptozotocin (STZ) in male Sprague-Dawley rats), (3) non insulin dependent diabetic rats (NIDDM; single subcutaneous injection of 90 mg/kg STZ in 5 day neonates), (4) tolbutamide-treated IDDM and (5) NIDDM (T-IDDM, T-NIDDM; giving tolbutamide 100 mg/kg/day for 6 weeks via an orogastric tube every day, respectively). At 14 weeks of age, experiments were performed using a Langendorff perfused heart preparation. After equilibration, T (myocardial developed tension), +dT/dt (contraction velocity), -dT/dt (relaxation velocity) and RT (resting tension) were measured during a 15 min period of global ischemia, followed by reperfusion for 20 min. Basal values of T increased in both T-IDDM and T-NIDDM, compared to IDDM and NIDDM, respectively. The percent recovery rate of +dT/dt in T-IDDM increased significantly during both ischemia and reperfusion, but the change in T-NIDDM was not significant. The recovery rates of -dT/dt in T-IDDM and T-NIDDM were significantly higher throughout reperfusion than in IDDM and NIDDM, respectively. On the other hand, that of T in T-IDDM and T-NIDDM were significantly higher than IDDM and NIDDM throughout ischemia and reperfusion, respectively. The RT was significantly higher in IDDM than in C and NIDDM throughout ischemia and reperfusion. The RT was significantly lower during ischemia in IDDM, but it did not differ significantly from IDDM during reperfusion. These results indicate that chronic oral administration of tolbutamide directly improved myocardial contractility throughout ischemia and reperfusion regardless of the improvement of glycemia. The improvement was also greater in IDDM than in NIDDM.     

Metabolism of pantothenic acid in hearts of diabetic rats

The metabolism of pantothenic acid (Pa) by cardiac muscle was studied in normal and diabetic rats. Tissue levels of Coenzyme A (CoA) are elevated in the heart during early (6 to 12 h) diabetes, remains at a high level for several days, and then returns to normal or below normal levels. The increase in total tissue CoA mainly occurs in myocytes as indicated by isolation of cardiac myocytes from control and diabetic animals and measuring their content of CoA. The CoA concentration increased from 37 to 93 microM in the cytosolic compartment and from 2.0 to 2.6 mM in the mitochondrial matrix. These effects of diabetes were reversed by insulin treatment. CoA synthesis in hearts removed from control rats and perfused in vitro was stimulated by including in the perfusate Pa, cysteine and dithiothreitol, but no exogenous energy substrate. This stimulated in vitro rate of CoA synthesis was reduced in hearts removed from diabetic animals, and the reduction increased with duration of diabetes. The reduced rate in diabetic hearts resulted from both a decreased rate of Pa phosphorylation and decreased Pa transport. Transport of Pa into myocytes was decreased by as much as 80% in hearts from diabetic animals. The low transport rate was due to a decrease in Vmax with no apparent change in Km. Treatment of the isolated heart with insulin did not correct the diabetic-induced reduction in Pa transport. The transport rate in normal and diabetic hearts was not influenced by the type of energy substrate provided to the heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

The metabolic response to norepinephrine in normal versus diabetic man

Summary Norepinephrine was infused for 60 minutes in high physiological concentration (0.08 μg/ kg/min) into seven insulin dependent diabetic subjects with no demonstrable endogenous insulin secretion and into seven normal subjects. Insulin dependent diabetic subjects had a stable, free insulin concentration of 23±5 μU/ml which was unaffected by norepinephrine infusion. In the normal subjects, norepinephrine induced an initial inhibition of insulin secretion which lasted for approximately 20 minutes. Norepinephrine infusion caused a rapid increase in both ketone body and glucose concentrations but this response did not differ between the two groups. In contrast, plasma nonesterified fatty acid and triglyceride concentrations were increased significantly more in the normal than in the diabetic subjects. The increase in plasma glucagon concentrations was similar in the two groups of subjects. The cause of the differential metabolic response to norepinephrine between the normal and diabetic groups was not resolved, but may be related, at least in part, to suppression of endogenous insulin secretion in the normal subjects.     

Effects of increased cardiac work on pyruvate dehydrogenase activity in hearts from diabetic animals

The effects of increased cardiac work and availability of pyruvate on the activation of pyruvate dehydrogenase (PDH) was studied in hearts isolated from diabetic rats. Diabetes resulted in complete inactivation of myocardial PDH. At low levels of cardiac work, PDH in hearts perfused with glucose or glucose plus insulin as substrate remained in the inactive form even after 25 min of in vitro perfusion indicating that the factors causing inactivation in the diabetic animal were not easily reversed in vitro. Raising the level of ventricular pressure development from 60 to 180 mmHg caused only a small increase in the percent of active PDH (from 0.3 to 16%). Comparable values in control hearts were 61 and 96% active PDH. Addition of high levels of perfusate pyruvate along with glucose increased the percent active PDH from 0.3 to 45 at 60 mmHg ventricular pressure. Although pyruvate increased active PDH the effect was much less than in normal hearts (85% active under comparable conditions). Increased ventricular pressure development (180 mmHg) in diabetic hearts receiving pyruvate caused a further activation of PDH to 66% but again this effect was much less than occurred in normal hearts (96% active).Inactivation of PDH in hearts from diabetic animals could not be accounted for by high mitochondrial levels of known effectors such as $\text{NADH}\text{NAD}$, acetyl $\text{CoA}\text{CoA}$ and $\text{ATP}\text{ADP}$. Increasing cardiac work resulted in decreased mitochondrial levels of NADH, acetyl CoA and ATP, but these changes had little effect on PDH activity. The data indicate that PDH in hearts of diabetic animals is resistant to activation by increased cardiac work and high tissue levels of pyruvate.