Jeanie M. Wood

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.

Inhibition of bovine heart Na+, K+-ATPase by palmitylcarnitine and palmityl-CoA.

Abstract The activity of a partially purified bovine heart Na+,K+-ATPase is inhibited by DL- and L- palmitylcarnitine (I50=44–48μM). Palmitylcarnitine with a I50 of 25μM also markedly inhibits K+-phosphatase activity. Palmityl-CoA decreases Na+,K+-ATPase activity, but to a lesser extent (I50=80μM). Both palmitic acid and hexanoic acid produce 10 to 15% inhibition of activity at concentrations of 70μM and 3–5mM, respectively. These free fatty acids protect the enzyme against inhibition by 40μM palmitylcarnitine. However, at 50μM palmitylcarnitine, the protective effect by hexanoic acid is no longer apparent. Addition of 40μM palmitylcarnitine to the Na+,K+-ATPase in the presence of varying concentrations of palmityl-CoA produces an additive inhibition of enzyme activity, suggesting two different sites on the enzyme susceptible to inhibition by the two ester forms of the fatty acid.

Palmitylcarnitine inhibition of the calcium pump in cardiac sarcoplasmic reticulum: A possible role in myocardial ischemia

Abstract Palmitylcarnitine is a time-dependent inhibitor of the Ca 2 + -ATPase activity of cardiac sarcoplasmic reticulum isolated from adult dogs. Half-maximal inhibition was obtained at approximately 20 μM (2 μmoles/mg). The extent of inhibition depended on the ratio of palmitylcarnitine to sarcoplasmic reticulum protein. Calcium uptake by cardiac sarcoplasmic reticulum (measured in the presence of sodium oxalate) was found to be even more sensitive to inhibition by palmitylcarnitine and complete inhibition was obtained at concentrations as low as 2.5 μM (0.25 μmole/mg) following preincubation. Calcium binding (measured in the absence of oxalate) was inhibited by palmitylcarnitine and calcium release was stimulated at similar ratios. The level of palmitylcarnitine has been reported to increase several fold in myocardial ischemia and inhibition of the sarcoplasmic reticulum calcium pump could conceivably contribute either to the initial loss of contractility or the subsequent inability to restore full contractile function after prolonged ischemia.

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.

Effect of palmitylcarnitine on ouabain binding to Na, K-ATPase.

Abstract Palmitylcarnitine, an endogenous long-chain fatty acyl ester, inhibited cardiac Na, K-ATPase activity and binding of [ 3 H]ouabain to the enzyme. The inhibitory effects on enzyme hydrolytic activity and drug binding were time and concentration dependent, but also dependent upon the ratio of palmitylcarnitine to protein. Palmitylcarnitine inhibitory effects were irreversible, but could be prevented by bovine serum albumin. In the presence of Mg 2+ + ATP or Mg 2+ + Pi, [ 3 H]ouabain binding was fully inhibited by 100 μ m palmitylcarnitine. The addition of sodium, or sodium plus potassium to the drug-binding medium reduced the inhibitory effect. The protective action of Na + was concentration dependent and was optimal at 75 μ m palmitylcarnitine. Equimolar amounts of choline chloride did not have the same protective effect as sodium chloride. Binding of ouabain to the enzyme in the absence of palmitylcarnitine prevented the protective effect of Na + . Inhibition of Na, K-ATPase functional properties occurred at a concentration range of palmitylcarnitine reported to occur in the cytosol of ischemic cells during episodes of experimental myocardial ischemia. It is suggested that elevated levels of palmitylcarnitine in ischemic myocardium may play a role in altering cellular function as well as the inotropic response of ischemic cardiac muscle to digitalis glycosides.

The effect of palmitoyl-coenzyme a on rat heart and liver mitochondria Oxygen consumption and palmitoylcarnitine formation

Rat heart and liver mitochondria, respectively, oxidized palmitoyl-CoA and palmitoylcarnitine optimally at 20-30 and 10-20 nmol substrate/mg. The oxidation of palmitoyl-CoA was accompanied by a lag in State 3 respiration that was proportional to the palmitoyl-CoA concentration. The delay in State 3 rates was more prolonged in liver than in heart at comparable palmitoyl-CoA levels. A similar range of palmitoyl-CoA concentrations produced significant inhibition of respiration in mitochondria oxidizing glutamate-malate. The inhibition was not due to a detergent effect of palmitoyl-CoA since addition of carnitine restored State 3 rates. Electron microscopic examination of mitochondria at low palmitoyl-CoA levels revealed normal ultrastructure. At comparable concentrations of palmitoyl-CoA, formation of palmitoylcarnitine by mitochondria from rat heart and liver followed first-order kinetics. The apparent first-order rate constants decreased with increasing palmitoyl-CoA. These results suggest that substrate inhibition may influence the rate of palmitoyl carnitine formation even at physiological concentrations of palmitoyl-CoA. The apparent first-order rate constant at palmitoyl-CoA levels (12 nmol palmitoyl CoA/mg) optimally oxidized by liver mitochondria, was one-third the value of the apparent rate constant measured in heart mitochondria at the identical substrate level. The prolongation in time to reach equilibrium may acocunt for the relatively greater respiratory sensitivity of liver mitochondria to increasing levels of palmitoyl-CoA.

Effects of glucagon, insulin and cyclic-AMP on mitochondrial calcium uptake in the liver

Abstract The effects of glucagon and insulin administration in vivo on hepatic mitochondrial Ca 2+ uptake were compared with the effects of these hormones when they were added directly to the perfused liver. Glucagon administration increased mitochondrial calcium uptake both in vivo and in the perfused liver. In contrast, while injection of insulin into rats stimulated, addition of insulin to the perfusate, inhibited Ca 2+ uptake. Cyclic AMP, when added to the perfusate, also increased the uptake of Ca 2+ by mitochondria, subsequently isolated. The possible implications of the results are discussed.

Effects of ouabain on calcium-45 flux in guinea pig cardiac tissue☆

Abstract Intact heart fragments (mince) from guinea pig were prepared by a mechanical chopper. Calcium-45 uptake in control and ouabain-treated tissue was measured after post-incubation of the tissue in a calcium-free medium containing lanthanum. After 30 min, ouabain significantly increased tissue calcium-45. In guinea pig heart mince loaded with calcium-45, the appearance of calcium-45 in the medium revealed an energy of activation of 8.2 kcal/mol; ouabain reduced this value 50%. Washout of calcium-45 in the presence or absence of ouabain was measured at 25°C. A portion of the washout appeared to be affected by ouabain; the T 1 2 for retention of tissue calcium-45 for this phase was increased 1.5 times. These results may bear on the mechanism of cardiac glycoside effects on the sodium gradient and its relationship to calcium movements.

Effect of malonyl-CoA on calcium uptake and pyridine nucleotide redox state in rat liver mitochondria.

A possible role for Ca*+ in the regulation of intracellular metabolic events has been proposed [l-3]. Pyruvate dehydrogenase phosphatase is stimulated [4] and pyruvate carboxylase inhibited [5] by mitochondrial Ca”. The effect of glucagon to increase gluconeogenesis and to stimulate ketogenesis is significantly diminished by cellular Ca*+ depletion [6]. In addition, malonyl-CoA concentrations decrease with glucagon treatment and increase in meal-fed rats [7-91. Recent evidence suggests that malonyl-CoA specifically inhibits liver mitochondrial carnitine palmitoyltransferase I [lo]. Palmitoyl-CoA has been shown to inhibit Ca*‘uptake and to effect Ca*+release from cardiac mitochondria [ 111. Possible regulation of Ca*+ flux by the redox state of mitochondrial pyridine nucleotides has also been suggested [ 121. In these studies, 4 PM palmitoyl-CoA inhibited Ca*+ uptake by rat liver mitochondria. When carnitine was added, Ca*+ uptake rates returned to normal, but addition of malonyl-CoA reduced the rates to levels seen with palmitoyl-CoA along (Ki 3 PM, malonyl-CoA). Ca’* release and pyridine nucleotide oxidation were induced by palmitoyl-CoA and these effects were modified by carnitine and malonyl-CoA. A possible regulation of mitochondrial Ca*+ movements by cellular lipid metabolites is suggested.

The effects of glucagon and epinephrine on two preparations of cardiac mitochondria

The effects of in vivo administration in epinephrine on calcium uptake were measured in two preparations of heart mitochondria, intermyofibrillar (IMF) and subsarcolemmal (SSL) using either 45Ca2+ or murexide to follow calcium movement. The administration of either hormones resulted in an increased calcium uptake in both preparation of mitochondria subsequently isolated. This increase might be the consequence of the increased State 3 respiration, also evoked by hormones. The possibility is raised that the inotropic actions of glucagon and epinephrine might be partially mediated by mitochondria.