Simultaneous evaluations of contractility and energy metabolism of stunned myocardium using 31P magnetic resonance spectroscopy.

A canine model of postischemic myocardial dysfunction (15 min ischemia, 60 min reperfusion) was used to evaluate the relationship between energy metabolism and myocardial contractile function by on-line measurements of ECG, left ventricular pressure, coronary blood flow and regional segment shortening (%SS) with the continuous acquisition of 31PMR spectra. Two groups emerged from these studies; the first (n = 7) in which regional myocardial %SS remained significantly depressed after 60 min of reperfusion (stunned) and the second (n = 5) in which regional %SS returned to control levels after 60 min of reperfusion (non-stunned). Both groups exhibited rapid, similar decreases in %SS and parallel rapid decreases in the phosphocreatine to inorganic phosphate (PCr/Pi) ratio with the onset of ischemia. The PCr/ATP ratio exceeded control levels in the stunned group immediately upon reperfusion and remained significantly above control after 60 min of reperfusion. Measurements of tissue myocardial creatine kinase (CK) revealed a significant decrease in total tissue CK activity in stunned myocardium compared to control. A significant inverse relationship (r = -0.904, p < 0.003) was found between myocardial tissue CK specific activity and the PCr/ATP ratios. We postulate that the elevated PCr/ATP ratio caused by the impairment of energy transfer to the contractile apparatus constitutes a contractile dysfunction in the postischemic heart.

Postischemic recovery of mitochondrial adenine nucleotides in the heart.

BACKGROUND: Adenine nucleotides (AdNs) are lost from the mitochondrial fraction of the heart cell during ischemia. It is unknown whether this pool of AdNs can be replenished after reperfusion. The purpose of this study was to evaluate the postischemic recovery of the mitochondrial AdN pool. METHODS AND RESULTS: The left anterior descending coronary artery (LAD) of the canine heart was occluded for 30 minutes followed by either no reflow, 30-minute reflow, 1-day reflow, or 7-day reflow. Systolic shortening in the LAD-supplied region was absent during occlusion but recovered to approximately 30% of preocclusion values during early reperfusion. Mitochondrial and tissue AdNs (ATP, ADP, and AMP) were determined in the LAD-supplied and left circumflex-supplied (control) regions of the heart. The AdN content (expressed as percent of control values) of mitochondria from the LAD region was 55 +/- 10% (p less than 0.002), 64 +/- 7% (p less than 0.001), 81 +/- 6% (p less than 0.03), and 94 +/- 8% for the no-reflow, 30-minute-reflow, 1-day-reflow, and 7-day-reflow groups, respectively. The AdN content (expressed as percent of control values) of tissue samples from the LAD region was 52 +/- 9% (p less than 0.002), 48 +/- 12% (p less than 0.02), 68 +/- 5% (p less than 0.002), and 70 +/- 9% for the no-reflow, 30-minute-reflow, 1-day-reflow, and 7-day-reflow groups, respectively. There was a good correlation between mitochondrial and tissue AdN (r = 0.95). Using initial exchange rates, adenine nucleotide translocase activities of mitochondria from the LAD and control regions were not significantly different. State 3 respiration of LAD mitochondria was depressed (approximately 25%, p less than 0.05) only in the no-reflow group. Acceptor control ratios of the LAD mitochondria were not significantly different from control values in any group. CONCLUSIONS: After 30 minutes of regional ischemia, postischemic restoration of the mitochondrial AdN pool occurs between 1 and 7 days; this restoration is preceded by recovery of respiratory and adenine nucleotide translocase functions. Although the abnormally low levels of AdN persist in the mitochondrial compartment during the early reperfusion period, postischemic contractile dysfunction cannot be explained by depressed mitochondrial respiratory activity.

In vitro and in vivo effects of R023-6152 on heart mitochondrial calcium and energy metabolism.

Previous studies have shown that Ca2+ channel antagonists in all chemical classes can inhibit Na(+)-induced CA2+ release from mitochondria. The effects of R023-6152, a new thiazepinone Ca2+ channel antagonist, on isolated rabbit heart mitochondrial Ca2+ transport and respiratory activity were compared with those of diltiazem. Heart mitochondria were also isolated and assayed from dogs treated in vivo with either R023-6152 or diltiazem. The results indicate that R023-6152 produces half-maximal inhibition of Na(+)-induced Ca2+ release from isolated mitochondria at relatively the same concentrations (10-30 microM) as diltiazem but also produces inhibition of mitochondrial Ca2+ uptake and state 3 respiration at concentrations (25-100 microM), at which diltiazem has no effect. The greater lipophilicity of R023-6152 in gaining access to and inhibiting the phosphate transporter in the mitochondrial membrane as compared with that of diltiazem may explain these results. Heart mitochondria isolated from dogs treated with diltiazem and R023-6152 exhibited lower rates of state 3 respiration as compared with controls. We suggest that this may result from a reduction in transsarcolemmal Ca2+ flux causing a down-regulation in mitochondrial dehydrogenase activity and not from any direct intracellular effects of the two drugs.

In vivo inhibition of platelet aggregation and occlusive coronary thrombus formation by a new calcium antagonist (R023-6152).

Reports have shown that all three major classes of calcium antagonists can inhibit platelet aggregation in vitro. In this study, we compared the platelet antiaggregatory effects of R023-6152, a thiazepinone-type calcium antagonist, with diltiazem in an in vivo canine model of spontaneous coronary thrombosis. Plasma serotonin (5-HT) levels measured in the coronary sinus were used as an index of in vivo platelet aggregation and coronary flow measured by a Doppler flow probe. Untreated controls developed total coronary occlusion in 62 +/- 18 min after the current used to initiate thrombus formation was discontinued. Control 5-HT levels peaked at 213 +/- 63 ng/ml just before occlusion. Dogs receiving intravenous (i.v.) R023-6152 (200 micrograms/kg) or diltiazem (50 micrograms/kg) immediately after the current was discontinued exhibited no significant elevations in 5-HT values (12.3 +/- 1.4 and 1.84 +/- 0.42 ng/ml for R023-6152 and diltiazem, respectively) or development of coronary occlusions in the next 3 h. Small, transient decreases in arterial pressure (8-10 mm Hg) and changes in contractility occurred during infusion of both drugs. Gravimetric determinations of thrombus weights showed significantly smaller thrombi in the drug-treated animals as compared with controls. The results indicate that both R023-6152 and diltiazem are effective in suppressing in vivo platelet aggregation associated with occlusive coronary thrombus formation.

Postnatal changes in heart mitochondrial calcium and energy metabolism.

The newborn mammalian heart has less functional capacity compared with the adult, yet newborn myocardial mitochondrial respiratory activity is the same or exceeds that of adult. This study was aimed at determining the temporal changes in newborn rabbit heart mitochondrial energy-linked Ca2+ transport during early postnatal development. At birth, substrate-supported Ca2+ uptake is twice that of adult and declines toward adult rates during the first 14 days. Both NADH- and succinate-linked respiration are equivalent to adult values at birth, increase transiently during the first 7 days, and then decline toward adult. Newborn heart mitochondrial preparations exhibit the same membrane potential (delta psi) values during Ca2+ uptake and have comparable rates of Na(+)-induced Ca2+ efflux as adult. Creatine kinase (CK) activity is very low in 1- to 7-day-old newborn mitochondria and increases rapidly toward adult values after 10 days of age. The decreasing rates of Ca2+ uptake do not appear to be related to respiratory activity, membrane potential, or increased cycling of Ca2+ but rather to a direct effect on the mitochondrial Ca2+ uniporter. Preliminary studies indicate changes in mitochondrial membrane phospholipids during early development that may be related to the increasing CK activity and decreasing Ca2+ uptake and respiration. We postulate that mitochondrial membrane lipid changes in early postnatal development may be the causative factor underlying these changes in functional activity.

Intermittent ischemia produces a cumulative depletion of mitochondrial adenine nucleotides in the isolated perfused rat heart.

The purpose of the present study was to determine if repetitive myocardial ischemia would result in the cumulative loss of mitochondrial adenine nucleotides. Isolated perfused rat hearts were subjected to continuous or intermittent ischemia. A single 5-minute period of continuous ischemia did not result in a significant decrease in the mitochondrial adenine nucleotide pool; a single 10-minute period of ischemia resulted in a decrease of approximately 17%. Next, the adenine nucleotide content of mitochondria from preischemic and 30-minute continuous ischemic hearts was compared with two groups of hearts undergoing intermittent ischemia (both groups receiving a total of 30 minutes of ischemia). One group received three 10-minute episodes of ischemia interrupted by 5-minute periods of reperfusion (3 x 10-minute intermittent ischemia); the other intermittent ischemic group received six 5-minute episodes of ischemia interrupted by 5-minute periods of perfusion (6 x 5-minute intermittent ischemia). The mitochondrial adenine nucleotide content (expressed as nanomoles per nanomole cytochrome a) for the preischemic and 30-minute continuous ischemic hearts was 14.7 +/- 0.6 and 8.0 +/- 0.4, respectively. The mitochondrial adenine nucleotide content of the 3 x 10-minute intermittent ischemia group (8.5 +/- 0.5) was not significantly different from the 30-minute continuous ischemic group. The mitochondrial adenine nucleotide content of the 6 x 5-minute intermittent ischemia group (11.0 +/- 0.6) was significantly larger than that of the 30-minute continuous and the 3 x 10-minute intermittent ischemia groups (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation of plasma serotonin changes with platelet aggregation in an in vivo dog model of spontaneous occlusive coronary thrombus formation.

The role of platelets in contributing to occlusive coronary artery thrombus formation remains unresolved. A large number of studies have utilized in vitro techniques to study platelet aggregation. This report describes a model of spontaneous in vivo thrombus formation which involves application of current in the left circumflex coronary artery of the dog. Changes in mean coronary blood flow velocity (50% above control) are used to predict the point at which current can be discontinued without interrupting the ongoing process of thrombus formation. Thrombus formation proceeds to total vessel occlusion within 62 +/- 18 minutes after discontinuation of current. Coronary sinus plasma serotonin concentrations are used as an in vivo index of platelet aggregation during thrombus formation. Plasma serotonin levels increased only slightly above baseline levels during initial thrombus formation. Coronary sinus serotonin levels rose markedly after cessation of current, reaching a peak just prior to total vessel occlusion. The marked increase in serotonin concentration observed in the latter stages of thrombus formation strongly suggests that platelet aggregation is a significant factor in the evolution of an occlusive coronary thrombus.

Amiloride and diltiazem inhibition of microsomal and mitochondrial Na+ and Ca2+ transport.

Amiloride, a K+-sparing diuretic, and diltiazem, a Ca2+ channel antagonist, have both been reported to inhibit Na+ transport-associated processes in different subcellular membrane systems. In this report, similar inhibitory effects of both agents are demonstrated on Na+-induced Ca2+ release from rabbit heart mitochondria and on Na+ uptake in a kidney medulla microsomal preparation. Both amiloride and diltiazem produce 50% inhibition of Na+ uptake in kidney microsomes at the same concentrations. Heart mitochondrial Na+-induced Ca2+ release was 50% inhibited by 6 microM diltiazem and 200 microM amiloride. No effects of either agent on mitochondrial respiratory activity were observed. The results suggest a specific effect of both drugs on a Na+-binding site associated with an antiport exchange process. These data also extend previous observations suggesting the use of these agents as tools to define further ion transport mechanisms in biological membranes.

Comparative functional properties of mitochondria from seal and dog hearts.

The harbor seal (Phoca vitulina) is capable of protracted dives resulting in low arterial PO2 levels. The mammalian heart is an aerobic organ depending primarily on mitochondrial oxidations for energy (ATP). Heart mitochondria were isolated from freshly killed seals and the functional data obtained compared to dog heart mitochondria isolated under similar conditions. The percentage yields of mitochondria based on cytochrome oxidase recovery were essentially the same from dog and seal hearts. However, the actual quantity of mitochondrial protein obtained per gram of seal heart tissue was lower than that isolated from dog heart. Phosphorylating rates of respiration (State 3; Q02) and cytochrome content were significantly lower in seal heart mitochondria compared to dog. The data indicate that seal hearts have fewer mitochondria per gram of tissue, lower active respiratory rates and lower cytochrome contents than dog heart.

Intramitochondrial adenine nucleotides and energy-linked functions of heart mitochondria.

Isolated rabbit heart mitochondria were incubated with varying amounts of inorganic pyrophosphate in 250 mM sucrose to specifically decrease the pool size of endogenous adenine nucleotides. The endogenous adenine nucleotide content decreased by as much as 80% as a result of this treatment. Phosphorylating respiration (state 3) declined from about 340 to 180 nAtoms O . min-1 . mg protein-1 over the full range of intramitochondrial adenine nucleotides measured (approx 7.5-1.5 nmol/mg protein). Uncoupled and nonphosphorylating (state 4) rates of respiration were not greatly affected by adenine nucleotide depletion. Respiratory activity of the adenine nucleotide-depleted mitochondria was enhanced by addition of exogenous adenosine 5'-triphosphate (ATP). Partial depletion (approx 40%) of the intramitochondrial adenine nucleotides resulted in an impaired ability of heart mitochondria to retain Ca2+. Premature Ca2+ efflux was associated with organelle swelling and altered energy coupling. Exogenous ATP or adenosine 5'-diphosphate (ADP) added prior to Ca2+ efflux restored Ca2+ retention in these mitochondria. Atractyloside inhibited the restoration of Ca2+ retention. This study indicates a significant role for endogenous adenine nucleotides in maintaining oxidative phosphorylation and Ca2+ transport in heart mitochondria. The results are discussed with regard to significance in ischemic heart damage.

Mechanism(s) of altered mitochondrial calcium transport in acutely ischemic canine hearts.

Studies were undertaken to determine the mechanisms leading to altered mitochondrial function in ischemic myocardium. A new procedure has been developed to routinely isolate 60-70% of the total mitochondrial protein from heart tissue. After 1 hour of ischemia, mitochondria exhibit decreases of more than 50% in phosphorylating respiration for both NADH- and succinate-linked substrates compared to controls. However, no significant decreases in the efficiency of mitochondrial ATP synthesis (ADP:0) or ATPase activity are observed. Rates of substrate-driven Ca2+ uptake exhibit decreases greater than that seen with phosphorylating respiration with incomplete uptake and premature release of Ca2+. Spectrophotometric measurements in ischemic heart reveal rapid oxidation or loss of mitochondrial NADH with marked "swelling" of the inner membrane compartment; both changes parallel the loss of Ca2+. Significant losses in intramitochondrial adenine nucleotides also are found. Mitochondrial retention of accumulated Ca2+ can be restored by addition of small amounts of exogenous adenine nucleotides (ATP or ADP) with concomitant attenuation of both NADH oxidation and "swelling." The data indicate that, following 1 hour of ischemia, the efficiency of mitochondrial ATP production is still relatively intact whereas both electron transport chain activity and calcium transport are severely compromised. These decreases appear to be related to selective membrane damage in the mitochondrial inner membrane.

Respiratory and calcium transport functions of mitochondria isolated from normal and transformed human lymphocytes.

Methods have been developed to isolate mitochondria from small amounts of normal and leukemic human lymphocytes obtained from small volumes of circulating blood. Mitochondrial respiratory functions were measured by polarographic techniques, and active calcium uptake was measured by a spectrophotometric procedure utilizing the calcium-sensitive dye murexide. The results indicate that the rates of active oxygen consumption of normal human lymphocyte mitochondria are very low in comparison to values obtained from a number of animal tissue sources. Mitochondria extracted from the lymphocytes of leukemic patients and from cultured, and phytohemagglutinin-transformed human lymphocytes exhibited higher respiratory rates than did normal controls. Although respiratory rates were relatively low in all preparations, the efficiency of energy coupling (ADP:O, ratio of nmol of adenosine diphosphate phosphorylated to nanoatoms of O2 consumed) was within normal limits. The mitochondria extracted from leukemia, cultured, and phytohemagglutinin-transformed lymphocytes exhibited high rates of respiratory substrate-supported calcium uptake compared to controls.

Role of mitochondria in heart cell function.

Mitochondrial oxidative phosphorylation constitutes the primary energy (ATP) supply in the myocardial cell. The principle substrates utilized in AtP synthesis are free fatty acids which must be converted to acylCoA esters prior to transport into the mitochondrial inner compartment. Although heart mitochondria are capable of rapidly accumulating significant amounts of calcium, their role in myocardial calcium metabolism is probably that of a "cytosolic buffer or reservoir" system. Energy-linked calcium transport and ATP synthesis compete for essentially the same "high-energy pool" in mitochondria. The integrated control of these processes remain to be elucidated. The acylCoA transferases, adenine translocase, CPK-MB enzyme system, Pi-OH exchanger and calcium influx and efflux channels located in the mitochondrial inner membrane all represent potential control points in the regulation of functional activity. Alterations in mitochondrial functions due to cardiac disease will not be fully understood until the fundamental mechanisms governing these processes in the normal myocardial cell are understood.

An improved method for purification of lymphocytes.

A method that combines glass-bead column filtration, Ficoll-Hypaque gradient separation, discontinuous sucrose gradient, and drastic reduction of cell transfers is described. The procedure gives a high yield of pure human lymphocytes from small amounts of blood, good preservation of B cell/T cell ratio, and sufficient material for subsequent biochemical studies.

Contractile function and myofibrillar ATPase activity in the exercise-trained dog heart.

Myocardial contractility and the enzymatic (ATPase) activity of cardiac contractile proteins were examined after exercise training using the chronically instrumented, unanesthetized dog as an experimental model. Before training, heart rate and the maximum rate of left ventricular pressure development (max dP/dt) were measured at rest and during submaximal exercise. Animals were then subjected to an 8- to 10-wk treadmill running program. Training was verified by the establishment of a 10- to 20-beat/min reduction in heart rate during submaximal exercise. After training max dP/dt was within normal limits at rest, but significantly elevated during submaximal exercise. When max dP/dt was plotted as a function of heart rate, either with the animal standing quietly on the treadmill or during submaximal exercise, a marked elevation in max dP/dt at any given heart rate was observed following training. Myofibrillar protein yield and ATPase activity values were nearly identical in left ventricles from exercise-trained and sedentary control dogs. Although exercise training by treadmill running improved contractile function in the unanesthetized dog myocardium, this response does not appear to involve alterations in myofibrillar ATPase activity.