Chylomicron metabolism by the isolated perfused mouse heart.

The catabolism of rat chylomicrons, labeled in their triacylglycerol (TG) component, was investigated using perfused working mouse hearts. Perfusion of mouse hearts with heparin increased lipoprotein lipase (LPL) activity in the perfusate. This heparin-releasable LPL pool remained constant over a variety of experimental conditions, including workload and fatty acid concentrations, making the mouse heart a suitable model to study chylomicron catabolism. Endothelium-bound LPL hydrolyzed radiolabeled (3)H-labeled chylomicrons (0.4 mM TG); the fate of LPL-derived (3)H-labeled fatty acids was split evenly between oxidation (production of (3)H(2)O) and esterification (incorporation into tissue lipids, mainly TG). In comparison, the oxidation of 0.4 mM [(3)H]palmitate complexed to albumin was fourfold greater than esterification into tissue lipids. Surprisingly, the addition of unlabeled palmitate (0.4 or 1.2 mM) to perfusions with (3)H-chylomicrons did not affect the fate (either oxidation or esterification) of LPL-derived (3)H-fatty acids. These results suggest that fatty acids produced from lipoprotein hydrolysis by the action of LPL and fatty acids from a fatty acid-albumin complex do not enter a common metabolic pool in the heart.

Glucose metabolism in perfused mouse hearts overexpressing human GLUT-4 glucose transporter.

Glucose and fatty acid metabolism was assessed in isolated working hearts from control C57BL/KsJ-m+/+db mice and transgenic mice overexpressing the human GLUT-4 glucose transporter (db/+-hGLUT-4). Heart rate, coronary flow, cardiac output, and cardiac power did not differ between control hearts and hearts overexpressing GLUT-4. Hearts overexpressing GLUT-4 had significantly higher rates of glucose uptake and glycolysis and higher levels of glycogen after perfusion than control hearts, but rates of glucose and palmitate oxidation were not different. Insulin (1 mU/ml) significantly increased glycogen levels in both groups. Insulin increased glycolysis in control hearts but not in GLUT-4 hearts, whereas glucose oxidation was increased by insulin in both groups. Therefore, GLUT-4 overexpression increases glycolysis, but not glucose oxidation, in the heart. Although control hearts responded to insulin with increased rates of glycolysis, the enhanced entry of glucose in the GLUT-4 hearts was already sufficient to maximally activate glycolysis under basal conditions such that insulin could not further stimulate the glycolytic rate.

Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice.

Contractile function and substrate metabolism were characterized in perfused hearts from genetically diabetic C57BL/KsJ-lepr(db)/lepr(db) (db/db) mice and their non-diabetic lean littermates. Contractility was assessed in working hearts by measuring left ventricular pressures and cardiac power. Rates of glycolysis, glucose oxidation, and fatty acid oxidation were measured using radiolabeled substrates ([5-(3)H]glucose, [U-(14)C]glucose, and [9,10-(3)H]palmitate) in the perfusate. Contractile dysfunction in db/db hearts was evident, with increased left ventricular end diastolic pressure and decreased left ventricular developed pressure, cardiac output, and cardiac power. The rate of glycolysis from exogenous glucose in diabetic hearts was 48% of control, whereas glucose oxidation was depressed to only 16% of control. In contrast, palmitate oxidation was increased twofold in db/db hearts. The hypothesis that altered metabolism plays a causative role in diabetes-induced contractile dysfunction was tested using perfused hearts from transgenic db/db mice that overexpress GLUT-4 glucose transporters. Both glucose metabolism and palmitate metabolism were normalized in hearts from db/db-human insulin-regulatable glucose transporter (hGLUT-4) hearts, as was contractile function. These findings strongly support a causative role of impaired metabolism in the cardiomyopathy observed in db/db diabetic hearts.

Voltage-sensitive dye mapping of activation and conduction in adult mouse hearts.

A custom-made apparatus based on a charge-coupled-device camera has been used to monitor changes in fluorescence from Langendorff-perfused adult mouse hearts stained with a voltage-sensitive dye, di-4-ANEPPS. With this approach it is possible to monitor activation of the ventricles at high temporal (375 micros/frame) and spatial resolution (72 x 78 pixels, 100 x 100 microm/pixel). In sinus rhythm, activation occurred with a complicated breakthrough pattern on both ventricles, and a total activation time of 3.51+/-0.16 ms (32 degrees C). A stimulus applied near the apex of the left ventricle resulted in a single activation wave front with a total activation time of 8.18+/-0.25 ms. Pacing from a site near the middle of the left ventricular epicardial surface revealed anisotropic conduction, indicating that conduction occurs preferentially in the direction of the predominant fiber orientation. The total activation time in this configuration was 5.44+/-0.24 ms. The difference in total activation time between sinus rhythm and epicardial stimulation suggests an important role for transmural conduction (the Purkinje system) in the mouse heart. These findings provide much of the necessary background needed for studying conduction abnormalities in genetically altered mice and suggest that the comparison of sinus rhythm and epicardial pacing can be used to reveal transmural conduction abnormalities.

Glucose and fatty acid metabolism in the isolated working mouse heart.

Although isolated perfused mouse heart models have been developed to study mechanical function, energy substrate metabolism has not been examined despite the expectation that the metabolic rate for a heart from a small mammal should be increased. Consequently, glucose utilization (glycolysis, oxidation) and fatty acid oxidation were measured in isolated working mouse hearts perfused with radiolabeled substrates, 11 mM glucose, and either 0.4 or 1.2 mM palmitate. Heart rate, coronary flow, cardiac output, and cardiac power did not differ significantly between hearts perfused at 0.4 or 1.2 mM palmitate. Although the absolute values obtained for glycolysis and glucose oxidation and fatty acid oxidation are significantly higher than those reported for rat hearts, the pattern of substrate metabolism in mouse hearts is similar to that observed in hearts from larger mammals. The metabolism of mouse hearts can be altered by fatty acid concentration in a manner similar to that observed in larger animals; increasing palmitate concentration altered the balance of substrate metabolism to increase overall energy derived from fatty acids from 64 to 92%.

The isolated working mouse heart: methodological considerations.

Our aim was to develop a working isolated murine heart model, as the extensive use of genetically engineered mice in cardiovascular research requires development of new miniaturized technology. Left ventricular (LV) function was assessed in the isolated working mouse heart perfused with recirculated oxygenated Krebs-Henseleit bicarbonate buffer (37 degrees C pH 7.4) containing 11.1 mM glucose and 0.4 mM palmitate bound to 3% albumin. The hearts worked against an afterload reservoir at a height equivalent to 50 mmHg, and heart rate was controlled by electrical pacing of the right atrium. LV pressure was measured with a micromanometer connected to a small steel cannula inserted through the apex of the heart. The experimental protocol consisted of two interventions. First, following instrumentation and stabilization, the preload reservoir was raised from a pressure equivalent of 7 to 22.5 mmHg, while pacing at 390 beats.min-1. Thereafter the height of the preload reservoir was set to 10 mmHg, and the pacing rate was varied from 260 to 600 beats.min-1. Aortic and coronary flows were measured by timed collections of effluent from the afterload line and that dripping from the heart, respectively [aortic+coronary flow=cardiac output (CO)]. Elevation of LV end-diastolic pressure (LVEDP) from approximately 5 to 10 mmHg resulted in a twofold increase in average cardiac power [product of LV developed pressure (LVDevP) and CO], whereas myocardial contractility (first derivative of LV pressure, dP/dt) and LVDevP (LV systolic pressure-LVEDP) increased only minimally (5-10%). Measured LVEDP was lower than the equivalent height of the preload reservoir by an amount that was related to the heart rate. Cardiac power, LVDevP and dP/dt were stable at heart rates up to 400 beats.min-1, but declined markedly with higher rates, consistent with the decrease in LVEDP. Thus, cardiac power was reduced to 50% of its maximum value when stimulated at approximately 500 beats.min-1, and at even higher rates there was little ejection. By systematic manipulation of the height of the preload reservoir and heart rate, we conclude that LV afterload and preload can be assessed only by high-fidelity measurement of intraventricular pressures. The heights of the afterload column and the preload reservoir are unreliable and potentially misleading indicators of LV afterload and preload.

Acetyl-CoA carboxylase control of fatty acid oxidation in hearts from hibernating Richardson's ground squirrels.

Although mammalian hibernators rely on stored body fat as a source of energy, direct measurement of energy substrate preference in heart tissue during hibernation, as well as potential mechanisms controlling fatty acid oxidation has not been examined. In order to determine whether an increase in fatty acid utilization occurs during hibernation, glucose and palmitate oxidation were measured in isolated working hearts from hibernating and non-hibernating Richardson's ground Squirrels. Hearts were perfused at either 37 degrees or 5 degrees C with perfusate containing 11 mM [U-14C]glucose and 1.2 mM [9,10-3H]palmitate, which allowed for direct measurement of both glucose oxidation (14CO2 production) and fatty acid oxidation (3H2O production). The contribution of fatty acid oxidation as a source of citric acid cycle acetyl-CoA was significantly greater in hearts from hibernating animals, compared to hearts from non-hibernating animals. Since acetyl-CoA carboxylase (ACC) regulates cardiac fatty acid oxidation (producing malonyl-CoA, a potent inhibitor of mitochondrial fatty acid uptake), we measured the activity and expression of ACC in these hearts. ACC activity was significantly decreased in hibernating ground squirrels, regardless of whether ACC was assayed at 37 degrees or 5 degrees C. This decrease in activity could not be explained by a change in the activity of 5'AMP-activated protein kinase, which can phosphorylate and inhibit ACC. Rather, the expression of the 280 kDa isoform of ACC (which predominates in cardiac muscle) was decreased in hearts from hibernating squirrel hearts. This suggests that a down regulation of ACC expression occurs as an adaptation for the increased utilization of fatty acid in hearts of hibernating ground squirrels.

Ca2+ uptake by cardiac sarcoplasmic reticulum at low temperature in rat and ground squirrel.

The Ca2+ uptake by isolated cardiac sarcoplasmic reticulum (SR) was compared between Richardson's ground squirrels and rats at 37, 25, 15, and 5 degrees C. The rate of SR Ca2+ uptake in ground squirrels was significantly higher than in rats over the temperature range. This marked species difference was observed over a Ca2+ concentration range from 0.1 to 10 microM. The Arrhenius plot for Ca2+ uptake was linear for ground squirrels between 37 and 5 degrees C but showed a depression from linearity for rats at 5 degrees C. This temperature sensitivity was also reflected in rat SR Ca2+-adenosinetriphosphatase activity. Analysis of [3H]ryanodine binding in SR suggests that more Ca2+ release channels are in an open state at low temperatures in rats than in ground squirrels. Together, these results suggest that species differences in the response of SR to low temperature may account for the rise in cytosolic free Ca2+ in cold-sensitive species and may be responsible, at least in part, for the inability of cold-sensitive hearts to function at low temperature.

Effects of hypothermia on energy metabolism in rat and Richardson's ground squirrel hearts.

Glycolysis, glucose oxidation, palmitate oxidation, and cardiac function were measured in isolated working hearts from ground squirrels and rats subjected to a hypothermia-rewarming protocol. Hearts were perfused initially for 30 min at 37 degrees C, followed by 2 h of hypothermic perfusion at 15 degrees C, after which hearts were rewarmed to 37 degrees C and further perfused for 30 min. Functional recovery in ground squirrel hearts was greater than in rat hearts after rewarming. Hypothermia-rewarming had a similar general effect on the various metabolic pathways in both species. Despite these similarities, total energy substrate metabolic rates were greater in rat than ground squirrel hearts during hypothermia despite a lower level of work being performed by the rat hearts, indicating that rat hearts are less efficient than ground squirrel hearts during hypothermia. After rewarming, energy substrate metabolism recovered completely in both species, although cardiac work remained depressed in rat hearts. The difference in functional recovery between rat and ground squirrel hearts after rewarming cannot be explained by general differences in energy substrate metabolism during hypothermia or after rewarming.

Effects of temperature and pH on cardiac myofilament Ca2+ sensitivity in rat and ground squirrel.

Chemically skinned papillary muscles from active and hibernating ground squirrels were used to determine whether the enhanced cardiac contractility observed in hibernation is due to a change in myofilament Ca2+ sensitivity. A similar preparation from rats was used to reflect the changes in a nonhibernator. When examined at pH 7.00 in all three groups and under physiological pH with varying temperatures in the ground squirrels, the calcium concentration at which muscle tension is at 50% maximum (pCa2+50) decreased significantly (P < 0.05) with decreasing temperature (25, 15, and 5 degrees C). When hibernating and active ground squirrels were compared, no significant difference in pCa2+50 was observed at 25 degrees C; however, the values at 15 and 5 degrees C were significantly higher (P < 0.05) in the hibernating squirrels. The results indicate that cardiac myofilament Ca2+ sensitivity decreases significantly at low temperature in both active and hibernating ground squirrels; however, the higher Ca2+ sensitivity in the hibernating squirrels at 15 and 5 degrees C could partially contribute to the enhanced cardiac contractility typically seen during hibernation.

Seasonal variations in the rate and capacity of cardiac SR calcium accumulation in a hibernating species.

The rate of calcium uptake and the level of calcium accumulation was measured in cardiac muscle SR from hibernating and nonhibernating Richardson's ground squirrels. In whole heart homogenates, the rate of calcium uptake was higher (P less than 0.05) in hibernating animals than it was in active animals. Further purification of homogenates into sacroplasmic reticulum (SR) preparations showed that the hibernating animals had the highest rate of calcium uptake and the greatest level of calcium accumulation. These results could not be explained by variations in non-SR membrane contaminants nor by changes in the maximal activity or total amount of a SR marker enzyme, the Ca(2+)-ATPase. The addition of ryanodine to the calcium uptake medium increased the level of calcium accumulation in all groups by a similar amount. It is concluded that the high rate of calcium uptake by isolated cardiac SR vesicles from hibernating ground squirrels reflects the activity of the organelle in vivo, and that the ability of the ryanodine-insensitive population of SR vesicles to accumulate calcium is affected by hibernation.

Effect of low temperature on the cytosolic free Ca2+ in rat ventricular myocytes.

The effect of low temperature on the cytosolic free Ca2+ [( Ca2+]i) has been investigated in isolated ventricular myocytes from adult rats using the fluorescent probe Indo-1. The distribution of Indo-1 between the mitochondrial and cytoplasmic compartments was first determined in the isolated myocytes using the digitonin and Triton X-100 treatments. By subtracting the mitochondrial [Ca2+]i from the total [Ca2+]i measured with Indo-1, the average cytosolic [Ca2+]i was found to increase significantly (P less than 0.05) from 139 nM to 255 and 297 nM when the temperature was decreased from 37 degrees C to 15 degrees and 5 degrees C, respectively. A marked increase in cytosolic [Ca2+]i to a new steady state level was observed when the membrane of myocytes was depolarized by 60 mM KCI; the average magnitude of increase being 110, 243 and 186 nM, at 37 degrees, 15 degrees and 5 degrees C respectively. Our results support the hypothesis that the cardiac arrhythmia typically observed in the hypothermic rat is due to an increased cytosolic [Ca2+]i with decreasing body temperature.