Palmitate oxidation by the mitochondria from volume-overloaded rat hearts.

In this work, an attempt was made to identify the reasons of impaired long-chain fatty acid utilization that was previously described in volume-overloaded rat hearts. The most significant data are the following: (1) The slowing down of long-chain fatty acid oxidation in severely hypertrophied hearts cannot be related to a feedback inhibition of carnitine palmitoyltransferase I from an excessive stimulation of glucose oxidation since, because of decreased tissue levels of L-carnitine, glucose oxidation also declines in volume-overloaded hearts. (2) While, in control hearts, the estimated intracellular concentrations of free carnitine are in the range of the respective Km of mitochondrial CPT I, a kinetic limitation of this enzyme could occur in hypertrophied hearts due to a 40% decrease in free carnitine. (3) The impaired palmitate oxidation persists upon the isolation of the mitochondria from these hearts even in presence of saturating concentrations of L-carnitine. In contrast, the rates of the conversion of both palmitoyl-CoA and palmitoylcarnitine into acetyl-CoA are unchanged. (4) The kinetic analyses of palmitoyl-CoA synthase and carnitine palmitoyltransferase I reactions do not reveal any differences between the two mitochondrial populations studied. On the other hand, the conversion of palmitate into palmitoylcarnitine proves to be substrate inhibited already at physiological concentrations of exogenous palmitate. The data presented in this work demonstrate that, during the development of severe cardiac hypertrophy, a fragilization of the mitochondrial outer membrane may occur. The functional integrity of this membrane seems to be further deteriorated by increasing concentrations of free fatty acids which gives rise to an impaired cooperation between palmitoyl-CoA synthase and carnitine palmitoyltransferase I. In intact myocardium, the utilization of the in situ generated palmitoyl-CoA can be further slowed down by decreased intracellular concentrations of free carnitine.

Control of oxidative metabolism in volume-overloaded rat hearts: effect of propionyl-L-carnitine.

The objective of the present work was the assessment of metabolic events responsible for the improvement of hemodynamic function of volume-overloaded hearts from rats receiving propionyl-L-carnitine. A severe cardiac hypertrophy was induced in 2-mo-old rats by surgical opening of an aortocaval communication. Three months later, during in vitro perfusions with 1.2 mM palmitate, 11 mM glucose, and 10 IU/l insulin, the mechanical performance and overall energy turnover (myocardial O2 consumption) of hypertrophied rat hearts were significantly decreased under conditions of moderate and high workloads. These changes in cardiac energetics paralleled the decrease in total tissue carnitine content and alterations in exogenous palmitate oxidation. The oxidative utilization of glucose was also slightly depressed in volume-overloaded hearts while steady-state glycolysis rates increased, especially in hearts subjected to high mechanical loads. This slowing of metabolic pathways involved in acetyl-CoA generation resulted in decreased NADH availability and in an apparent substrate limitation of oxidative phosphorylation suggested by a failure of cytosolic unbound ADP to drive respiration. Long-term administration of propionyl-L-carnitine normalized the degree of reduction of mitochondrial pyridine nucleotides and improved the kinetics of mitochondrial ATP production in volume-overloaded hearts. The resulting acceleration of energy turnover was essentially related to improved oxidative utilization of glucose, but steady-state palmitate oxidation rates also increased in severely hypertrophied hearts. This concomitant acceleration of glucose and palmitate oxidation may be related to the particular experimental conditions (high exogenous palmitate concentrations, elevated workloads) used in this study. We assume that the increase in intracellular carnitine, together with a stimulation of acetyl-CoA demands related to high workloads, creates conditions that are compatible with the simultaneous relief of pyruvate dehydrogenase and carnitine palmitoyltransferase I. The resulting increase in the rate of steady-state ATP production improves, in turn, the mechanical activity of volume-overloaded hearts.

Uncoupling of contractile function from mitochondrial TCA cycle activity and MVO2 during reperfusion of ischemic hearts.

In this study we determined whether contractile function becomes uncoupled during reperfusion of ischemic hearts from mitochondrial tricarboxylic acid (TCA) cycle activity or myocardial O2 consumption (MVO2). Isolated working rat hearts perfused with buffer containing 1.2 mM palmitate and 11 mM glucose were subjected to 30 min of global ischemia followed by 60 min of aerobic reperfusion. During reperfusion, cardiac work recovered to 26.5 +/- 5.4% (n = 29) of preischemic levels, even though TCA cycle activity, fatty acid beta-oxidation, glucose oxidation, glycolysis, and MVO2 rapidly recovered. As a result, the efficiency of coupling between cardiac work and TCA cycle activity and between cardiac work and mitochondrial respiration decreased during reperfusion. In contrast, coupling of TCA cycle activity to MVO2 during reperfusion recovered to preischemic values. Addition of 1 mM dichloroacetate at reperfusion resulted in a significant increase in both cardiac work and cardiac efficiency during reperfusion. This was associated with a significant decrease in H+ production due to an improved balance between glycolysis and glucose oxidation. These data demonstrate that mitochondrial function and overall myocardial ATP production quickly recover in rat hearts after a 30-min period of global ischemia. However, mitochondrial ATP production is not efficiently translated into mechanical work during reperfusion. This may be due to an imbalance between glycolysis and glucose oxidation, resulting in an increase in H+ production and a decrease in cardiac efficiency.

Assessment of the cardiostimulant action of propionyl-L-carnitine on chronically volume-overloaded rat hearts.

Chronic volume overload was induced in young rats of Wistar strain by surgical opening of the aorto-caval fistula. Three months later, during in vitro perfusion with exogenous palmitate, left ventricular function and energy turnover (QO2) of hypertrophied hearts were severely depressed. This seemed to be related to impaired long-chain fatty acid utilization, as reflected by decreased 14CO2 production from U-14C-palmitate and decreased tissue levels of L-carnitine. Another group of rats exposed to chronic volume overload was pretreated for 2 weeks before sacrifice with propionyl-L-carnitine (250 mg/kg/day), and the hearts were perfused with 1.2 mM palmitate and 10 mM propionyl-L-carnitine. In this group, both mechanical performance and the oxygen consumption rate were quite comparable to those of untreated controls. On the other hand, tissue levels of L-carnitine were only slightly increased, and the rate of 14CO2 production from U-14C-palmitate was insignificantly improved. This suggests that propionyl-L-carnitine administration promotes the mechanical performance of normoxic volume-overloaded hearts via a mechanism other than improved palmitate utilization. The possibility that propionyl moieties themselves replenish with mitochondrial intermediates of the tricarboxylic cycle (malate, acetyl-CoA) is not excluded.

Fatty acid oxidation and mechanical performance of volume-overloaded rat hearts.

Chronic volume overload was induced in 2-mo-old rats by surgical opening of the aortocaval fistula. Rats were killed 3 mo later and their hearts were atrially perfused. During the perfusions with 1.2 mM palmitate, mechanical performance of volume-overloaded hearts was significantly decreased both under conditions of a moderate work load and, mainly, after the clamp of the aortic outflow line. Respective O2 consumption rates as well as the rates of 14CO2 production from [U-14C]palmitate were decreased to the same extent. When 2.4 mM octanoate was used as the exogenous substrate, both the O2 consumption rates and the rates of CO2 production of volume-overloaded hearts became comparable to those of control hearts perfused with same substrate. Mechanical activity of volume-overloaded hearts returned to control values and remained stable during the entire perfusion period tested. Total tissue L-carnitine was decreased by approximately 30% in volume-overloaded hearts, which may suggest that palmitate oxidation has been limited at the level of carnitine-acylcarnitine translocase. However, our polarographic studies of the respiratory activity of isolated mitochondria indicated that the palmitoylcarnitine translocation proceeds normally. On the other hand, state 3 respiration of the mitochondria from volume-overloaded hearts supplemented with either palmitate or palmitate and L-carnitine was significantly lower than that of control ones. This may suggest that an alteration of the enzymes involved in long-chain fatty acid activation and/or long-chain fatty acyl transfer to L-carnitine has developed under conditions of chronic mechanical overloading of the heart.

Carnitine transport and exogenous palmitate oxidation in chronically volume-overloaded rat hearts.

L-Carnitine transport and free fatty acid oxidation have been studied in hearts of rats with 3-month-old aorto-caval fistula. For carnitine transport experiments, the hearts were perfused via the ascending aorta with a bicarbonate buffer containing 11 mM glucose and variable concentrations L-[14C]carnitine (10-200 microM). In some experiments, the active component of carnitine transport was suppressed by the adjunction of 0.05 mM mersalyl acid. The subtraction of passive from total transport allowed reconstruction of the saturation curves of the carrier-mediated transport of L-carnitine. Our data suggest that at a physiological carnitine concentration (50 microM), the rate of [14C]carnitine accumulation was significantly depressed in mechanically overloaded hearts. In addition, according to Lineweaver-Burk analysis, the affinity of the membrane carrier for L-carnitine was considerably diminished (Km carnitine 125 instead of 83 microM, Vmax unchanged). The above alterations of L-carnitine transport did not result from a decrease of the transmembrane gradient of sodium, since the intracellular Na+ content of the hypertrophied hearts was quite similar to that of control hearts. The ability of atrially perfused, working hearts to oxidize the exogenous free fatty acids was assessed from 14CO2 production obtained in the presence of [U-14C]palmitate or [1-14C]octanoate. The total 14CO2 production, expressed per min per g dry weight, was significantly diminished in hearts from rats with the aorto-caval fistula if 1.2 mM palmitate was used. On the other hand, in the presence of 2.4 mM octanoate, a substrate which circumvents the carnitine-acylcarnitine translocase, no such reduction of the 14CO2 production could be detected. Our results suggest that the decrease of L-carnitine transport, resulting in a significant depression of tissue carnitine, may impair long-chain fatty acid activation and/or translocation into mitochondria. In contrast, the oxidation of short-chain fatty acids, the activation of which takes place directly in mitochondrial matrix, is not limited in volume-overloaded hearts.

Decreased L-carnitine transport in mechanically overloaded rat hearts.

The transport of L[14C] carnitine was studied in rat hearts with a three-month-old aorto-caval fistula. Tissue TG content was determined in order to assess the state of FFA utilization. The hearts were perfused with a bicarbonate buffer containing 11 mM glucose and variable concentrations (10-200 microM) of L[14C] carnitine. In some experiments, the active component of carnitine transport was suppressed by the adjunction of 0.05 mM mersalyl acid. The subtraction of passive from total transport allowed us to reconstruct the saturation curves of the net active transport of L-carnitine. Our results suggest that at physiological carnitine concentration (50 microM) the uptake of L-carnitine is significantly depressed in mechanically overloaded hearts. These changes are not related to alterations of coronary perfusion, since coronary flow rates (ml/min/g dry wt) are quite comparable in both groups tested. According to the Lineweaver-Burk analysis of the kinetics of saturable transport, the affinity of the membrane carrier for L-carnitine is considerably diminished in the overloaded hearts (Km[carnitine] 125 instead of 83 microM). The alterations of the kinetics of carnitine transport do not seems to be related to the decrease of the transmembrane gradient of sodium: the intracellular sodium content of the hypertrophied, but non-failing, hearts is quite similar to that of control hearts. In addition, carnitine deficiency does not lead to TG accumulation, at least under in situ conditions.