Contribution of shrinkage of extracellular space to extracellular K+ accumulation in myocardial ischaemia of the rabbit.

1. The contribution of the concentrating effect due to shrinkage of the extracellular space (ECS) to cellular K+ efflux on extracellular potassium ([K+]o) accumulation in response to ischaemia was investigated in an isolated, blood-perfused rabbit papillary muscle preparation with a confined extracellular space. 2. The ECS was quantified using either of two extracellular markers, choline or tetramethyl ammonium (TMA), each with specific ion-selective electrodes, as well as by measurement of extracellular resistance (ro). [K+]o and [Na+]o were also measured simultaneously using K(+)- and Na(+)-selective electrodes. 3. During ischaemia, [K+]o increased 3-fold from 4.2 +/- 0.1 to 12.6 +/- 1.0 mM at 10 min (n = 10) analogous to changes in the ischaemic heart in vivo. The ECS decreased to 83.9 +/- 3.2% of control measured using 1 mM choline extracellularly (n = 9, P < 0.01) or to 85.7 +/- 0.7% of control using 1 mM TMA (n = 6, P < 0.01). Nearly identical decreases in ro (84.1 +/- 2.4%, n = 15, P < 0.01) occurred simultaneously. 4. The small decrease in the ECS contributed only 0.8-0.9 mM to the total increase in [K+]o of 8.4 mM and had a minor effect on transmembrane K+ flux. No significant differences between the relative changes in [choline] and [Na+]o were observed. This excluded a major transmembrane Na+ movement during early ischaemia. 5. Bumetanide (10 mM), an inhibitor of K(+)-Cl- cotransport, a process which is involved in cell volume regulation consequent to osmotic cell swelling, significantly attenuated the increase in [K+]o after 6 min of ischaemia (8.3 +/- 0.6 mM, n = 5 vs. 10.3 +/- 0.4 mM in the control group, n = 6, P < 0.05), whereas N-ethylmaleimide (1 mM), a stimulator of this cotransporter, augmented [K+]o accumulation (12.0 +/- 0.6 mM at 6 min, P < 0.05). 6. We conclude that during early myocardial ischaemia, a major component of [K+]o accumulation is not caused by diminution of ECS per se, but rather by increased net K+ efflux due in part to K(+)-Cl cotransport secondary to myocyte volume regulation.

Deleterious effects of a systemic lytic state on reperfused myocardium. Minimization of reperfusion injury and enhanced recovery of myocardial function by direct angioplasty.

BACKGROUND: The beneficial effects of flow restoration and the deleterious impact of reperfusion injury on ischemic myocardium are well known. However, most experimental studies have induced reperfusion by mechanical release of nonthrombotic occlusions, only occasionally in the presence of a systemic lytic state. Conditions differ markedly in patients undergoing pharmacological or mechanical recanalization of thrombotically occluded coronary arteries. Accordingly, this study was designed to determine whether the method of coronary occlusion and mode of recanalization influence the response of the heart to reperfusion. METHODS AND RESULTS: The acute effects of reperfusion on right ventricular (RV) function and histology were studied in open-chest dogs subjected to right coronary artery (RCA) balloon occlusion and deflation alone (group 1), pharmacological lysis of thrombotic occlusions (group 2), balloon occlusion with reperfusion induced by balloon deflation in the presence of a systemic lytic state (group 3), and recanalization of thrombotically occluded vessels by direct angioplasty (group 4). In all groups, 1 hour of RCA occlusion led to RV free wall (FW) dyskinesis. In group 1, reperfusion promptly improved RVFW function, with normal RVFW thickness and only minimal edema by microscopy. In contrast, in group 2, clot lysis led to acute RVFW swelling and impaired recovery of RVFW contraction associated with striking interstitial edema, contraction band necrosis, and hemorrhage by microscopy. In group 3, balloon deflation in the presence of a lytic state led to a similar but less severe pattern of abrupt RVFW swelling and impaired recovery of RVFW function but lesser histological alterations than in group 2. However, mechanical recanalization of thrombotically occluded vessels (group 4) led to prompt recovery of RVFW function without significant RVFW swelling or histological abnormalities. CONCLUSIONS: Our observations indicate that the responses of ischemic myocardium to reperfusion are influenced by factors beyond those effects attributable to ischemia and reperfusion per se. Pharmacological lysis of coronary thrombi results in alterations characteristic of reperfusion injury and associated with impaired functional recovery. Such changes are also evident, although to a lesser extent, when reperfusion of northrombotic occlusions is induced by mechanical recanalization in the presence of a systemic lytic state but not in its absence. However, such effects were not seen with direct mechanical recanalization of thrombotically occluded vessels. In aggregate, these findings indicate that induction of a systemic lytic state, together with products released by lysis of intracoronary thrombi, generates an inurious milieu that exerts adverse effects on reperfused myocardium.

Palmitoylcarnitine increases [Na+]i and initiates transient inward current in adult ventricular myocytes.

This study was performed to determine whether long-chain acylcarnitines, specifically palmitoylcarnitine, could account for the increase in intracellular Na+ ([Na+]i) during ischemia eliciting a secondary increase in intracellular Ca2+ ([Ca2+]i). Accordingly, whole cell voltage-clamp procedures and Na(+)-sensitive electrode recordings were employed simultaneously in isolated adult rabbit ventricular myocytes to assess the relationship between activation of a slow-inactivating Na+ current [INa(s)] and a potential increase in [Na+]i. The [Na+]i increased progressively from 8.4 +/- 1.2 to 22.5 +/- 1.8 mM (n = 8, P < 0.01) on exposure to palmitoylcarnitine (10 microM) accompanied by the activation of INa(s); both effects were reversible. Inhibition of INa(s) by tetrodotoxin (TTX, 10 microM) inhibited the increase in [Na+]i. Increasing [Na+]i to 20 mM without ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to mimic effects measured with palmitoylcarnitine consistently elicited the transient inward current (Iti) and delayed afterdepolarizations (DADs). The percent inhibition (12.9 +/- 2.8%) of the Na(+)-K(+)-adenosinetriphosphatase pump activity by palmitoylcarnitine (10 microM) was much smaller than that induced by ouabain (10 microM, 90.5 +/- 2.5%), suggesting that this modest effect of palmitoylcarnitine on the pump is unlikely to account for the increase in [Na+]i induced by palmitoylcarnitine. Thus palmitoylcarnitine induces the INa(s) leading to an increase in [Na+]i, which elicits an increase in [Ca2+]i probably via the Na+/Ca2+ exchanger, thereby leading to the development of Iti and DADs.

Selected metabolic alterations in the ischemic heart and their contributions to arrhythmogenesis.

Myocardial ischemia in vivo is associated with dramatic electrophysiologic alterations which occur within minutes of cessation of coronary flow and are rapidly reversible with reperfusion. This suggests that subtle and reversible biochemical and/or ionic alterations within or near the sarcolemma may contribute to the electrophysiologic derangements. Our studies have concentrated on 2 amphipathic metabolites, long-chain acylcarnitines and lysophosphatidylcholine (LPC) which have been shown to increase rapidly in ischemic tissue in vivo and to elicit electrophysiologic derangements in normoxic tissue in vitro. Incorporation of these amphiphiles into the sarcolemma at concentrations of 1 to 2 mol%, elicits profound electrophysiologic derangements analogous to those observed in ischemic myocardium in vivo. LPC is produced in endothelial cells and myocytes in response to thrombin. Thus, activation of the coagulation system during ischemia may result in extracellular production and accumulation of LPC. The pathophysiological effects of the accumulation of both amphiphiles are thought to be mediated by alterations in the biophysical properties of the sarcolemmal membrane, although there is a possibility of a direct effect on ion channels. Inhibition of carnitine acyltransferase I in the ischemic cat heart was found to prevent the increase in both long-chain acylcarnitines and LPC and to significantly reduce the incidence of malignant arrhythmias including ventricular tachycardia and fibrillation. This review focuses on the influence of these amphiphiles on cardiac ionic currents observed during early ischemia and presents data supporting the concept that accumulation of these amphiphiles within the sarcolemma contributes to changes in ionic conductances leading to electrophysiological derangements. The contribution and the accumulation of these amphiphiles to alterations in intracellular Ca2+ as related to changes in Na/K-ATPase activity and intracellular Na+ are examined. Other alterations occur during early myocardial ischemia in addition to the events reviewed here; however, the results of multiple studies over the past 2 decades indicate that accumulation of these amphiphiles contributes importantly to arrhythmogenesis and that development of specific inhibitors of carnitine acyltransferase I or phospholipase A2 may be a promising therapeutic strategy to attenuate the incidence of lethal arrhythmias associated with ischemic heart disease in man.

Activation of thrombin receptor increases intracellular Na+ during myocardial ischemia.

Recent evidence indicates that factors involved in the activation of the coagulation system eliciting an intracoronary thrombus may contribute importantly to arrhythmogenesis during acute myocardial ischemia. In the present study, the influence of the thrombin receptor activating peptide, SFLLRNPNDKYEPF (SFLL), on intracellular Na+ ([Na+]i) and tissue lysophosphatidylcholine (LPC) content during ischemia was investigated in an isolated, blood-perfused rabbit papillary muscle preparation. During normoxic perfusion, [Na+]i, determined by an intracellular Na(+)-selective electrode, was 11.6 +/- 0.2 mM (n = 14) in the presence of a physiological concentration of LPC (125 microM) bound to albumin (control group). The addition of SFLL (100 microM, n = 12) to the LPC-containing perfusate had no significant additional effects on [Na+]i, twitch tension, action potential duration, or tissue LPC content. During zero flow ischemia, [Na+]i in the control group rose to 15.5 +/- 1.4 mM (P < 0.05) at 6 min, whereas [Na+]i in the group treated with SFLL increased rapidly from the preischemic value of 11.7 +/- 0.3 to 23.5 +/- 1.9 mM (P < 0.01 compared with that in the control group) over the same time period. This rapid rise in [Na+]i was associated with a greater accumulation of tissue LPC, an arrhythmogenic lipid metabolite, and the development of early ventricular arrhythmias. These results indicate that an increase in [Na+]i induced by activation of the thrombin receptor, likely mediated through its effect on the accumulation of LPC within ischemic myocardium, may be responsible for arrhythmogenesis during myocardial ischemia secondary to activation of the coagulation system.

Electrophysiologic basis, surgical development, and clinical results of the maze procedure for atrial flutter and atrial fibrillation.

After more than a decade of experimental and clinical research into the basic mechanisms underlying atrial fibrillation, we were able to develop a surgical procedure that appears to cure the arrhythmia. This surgical procedure has been used in 100 patients in our institution and in a total of approximately 130 patients by surgeons in other institutions. The surgical results have been excellent, which indicates the sophisticated electrophysiologic mapping systems are unnecessary and that the results are not surgeon-specific.

Biochemical and electrophysiological alterations underlying ventricular arrhythmias in the failing heart.

Our understanding of the electrophysiological and biochemical mechanisms underlying malignant ventricular arrhythmias in the setting of heart failure has been limited, in large part because of the lack of experimental preparations of heart failure that demonstrate spontaneously occurring ventricular arrhythmias. Recent 3-dimensional cardiac mapping studies in experimental preparations of heart failure, as well as the failing human heart, have demonstrated that focal non-reentrant mechanisms may underlie ventricular tachycardia occurring spontaneously or induced by programmed electrical stimulation. This non-reentrant activation may be due to triggered activity arising from delayed after depolarization. Alterations of calcium homeostasis in the failing heart involving a number of ionic channels and membrane transporters may contribute to increased levels of intracellular calcium and the activation of a transient inward current. Modulation of calcium flux by alpha- and beta-adrenergic stimulation may impact significantly on development of arrhythmias in the failing heart. Activation of the renin-angiotensin system and the generation of free radicals may also contribute. A thorough understanding of the underlying electrophysiological and biochemical alterations responsible for arrhythmogenesis in the failing heart will be critical for the development of therapeutic agents to prevent sudden death in patients with congestive heart failure.

Phosphatidic acid increases in response to noradrenaline and endothelin-1 in adult rabbit ventricular myocytes.

OBJECTIVE: The aim was to assess whether noradrenaline and endothelin-1 can stimulate endogenous production of phosphatidic acid in adult ventricular myocytes. METHODS: After stimulation of rabbit ventricular myocytes with noradrenaline and endothelin-1, total lipids were extracted using the Bligh and Dyer procedure and separated by thin layer chromatography, and phosphatidic acid was quantified using photodensitometric analysis of visualised lipids with CuSO4/H3PO4. RESULTS: Noradrenaline (10(-5) M) elicited a rapid increase in phosphatidic acid at 2 min, followed by a decrease at 5 min. A second delayed and sustained increase in phosphatidic acid occurred at 10 min. The response to noradrenaline (10(-9) to 10(-5) M) was concentration dependent with a half maximum response (EC50) of 3.1 x 10(-8) M and the maximum effect at 10(-6) M. The increase in phosphatidic acid production in response to noradrenaline was abolished by an alpha 1 adrenergic receptor blocking agent (2-[beta-(4-hydroxyphenyl)-ethylaminomethyl]tetralone) but unaffected by the beta adrenergic blocking agent L-propranolol. An increase in phosphatidic acid was also elicited in rabbit ventricular myocytes in response to endothelin-1. The response was time and concentration dependent with the maximal increase at 12 min, EC50 5.3 x 10(-9) M, and maximum effect at 10(-6) M. Both noradrenalin and endothelin-1 stimulated phosphatidylbutanol production in the presence of butanol (100 mM), indicating that both agonists activate phospholipase D. CONCLUSIONS: Noradrenaline at physiological concentrations elicits both a rapid and a delayed increase in phosphatidic acid in adult rabbit ventricular myocytes. Endothelial-1, at physiological concentrations, also stimulates an increase in the mass of phosphatidic acid in myocytes, but the increase induced by endothelin-1 is monophasic, in contrast to the biphasic response seen during stimulation with noradrenaline. Activation of phospholipase D contributes to the increase in phosphatidic acid seen during stimulation of myocytes with either noradrenaline or endothelin-1. These are the first data to characterise endogenous production of phosphatidic acid in isolated adult ventricular myocytes.

Neural dysfunction and metabolic imbalances in diabetic rats. Prevention by acetyl-L-carnitine.

The rationale for these experiments is that administration of L-carnitine and/or short-chain acylcarnitines attenuates myocardial dysfunction 1) in hearts from diabetic animals (in which L-carnitine levels are decreased); 2) induced by ischemia-reperfusion in hearts from nondiabetic animals; and 3) in nondiabetic humans with ischemic heart disease. The objective of these studies was to investigate whether imbalances in carnitine metabolism play a role in the pathogenesis of diabetic peripheral neuropathy. The major findings in rats with streptozotocin-induced diabetes of 4-6 weeks duration were that 24-h urinary carnitine excretion was increased approximately twofold and L-carnitine levels were decreased in plasma (46%) and sciatic nerve endoneurium (31%). These changes in carnitine levels/excretion were associated with decreased caudal nerve conduction velocity (10-15%) and sciatic nerve changes in Na(+)-K(+)-ATPase activity (decreased 50%), Mg(2+)-ATPase (decreased 65%), 1,2-diacyl-sn-glycerol (DAG) (decreased 40%), vascular albumin permeation (increased 60%), and blood flow (increased 65%). Treatment with acetyl-L-carnitine normalized plasma and endoneurial L-carnitine levels and prevented all of these metabolic and functional changes except the increased blood flow, which was unaffected, and the reduction in DAG, which decreased another 40%. In conclusion, these observations 1) demonstrate a link between imbalances in carnitine metabolism and several metabolic and functional abnormalities associated with diabetic polyneuropathy and 2) indicate that decreased sciatic nerve endoneurial ATPase activity (ouabain-sensitive and insensitive) in this model of diabetes is associated with decreased DAG.

Increased lysophosphatidylcholine content induced by thrombin receptor stimulation in adult rabbit cardiac ventricular myocytes.

OBJECTIVE: The aims were (1) to determine whether thrombin, which is increased in the presence of coronary thrombosis, can directly stimulate the production of lysophosphatidylcholine, which has arrhythmogenic properties, in ventricular myocytes; (2) whether the effect is dependent upon extracellular [Ca2+]; and (3) whether it is mediated directly through stimulation of the thrombin receptor. METHODS: Lipids were extracted from isolated adult rabbit ventricular myocytes and lysophosphatidylcholine was isolated by HPLC and quantified using a recently developed radiometric assay employing 3H-acetic anhydride. RESULTS: Thrombin (0.05 U.ml-1) stimulation of ventricular myocytes resulted in a nearly sixfold increase in lysophosphatidylcholine levels [0.26(SEM 0.03) to 1.61(0.42) nmol.mg-1 protein] within 1 min. The increase in myocytic lysophosphatidylcholine content was prevented by preincubation of thrombin with the proteolytic site inhibitors phenyly-prolyl-arginyl-chloromethyl ketone (PPACK) and dansylarginine N-(3-ethyl-1,5-pentanediyl)amide. The increase in lysophosphatidylcholine content in response to thrombin was not present at an extracellular calcium concentration ([Ca2+)]o) = 500 microM, but was marked at a physiological level of [Ca2+]o = 1.8 mM. Stimulation of myocytes with the thrombin receptor activating peptide SFLLRNPNDKYEPF (100 microM for 1 min) resulted in a similar increase in lysophosphatidylcholine content [1.61(0.27) nmol.mg-1 protein]. CONCLUSIONS: The marked increase in lysophosphatidylcholine content in cardiac myocytes in response to thrombin has important implications as an arrhythmogenic mechanism during early myocardial ischaemia.

Arrhythmogenic influence of intracoronary thrombosis during acute myocardial ischemia.

BACKGROUND: Patients with acute coronary artery thrombosis often develop primary malignant ventricular arrhythmias (MVA) early after coronary occlusion. In contrast, acute ischemia induced by nonthrombotic balloon occlusion during routine coronary angioplasty rarely elicits such arrhythmias. This study was designed to assess the role of intracoronary thrombosis in arrhythmogenesis during acute ischemia. METHODS AND RESULTS: We compared the incidence of MVA associated with acute left anterior descending coronary artery (LAD) thrombosis elicited in open-chest anesthetized dogs by electrical injury (n = 10) or intracoronary stent (n = 9) versus LAD balloon occlusion (n = 15). Compared with animals subjected to balloon occlusion, those with thrombotic occlusion had a significantly greater incidence of MVA, defined as nonsustained ventricular tachycardia (total duration > 10 seconds), sustained ventricular tachycardia, or ventricular fibrillation developing within the first 30 minutes of occlusion. In the combined thrombosis groups, MVA developed in 11 of 19 animals (58%) (6 of 10 dogs with electrical injury and 5 of 9 stent animals). In contrast, MVA occurred in only 1 of 15 animals (7%) subjected to balloon occlusion. This striking and significant difference in arrhythmias occurred despite the fact that radioactive microsphere perfusion analysis documented that the extent of left ventricular myocardium rendered ischemic was equal in all groups (percent of left ventricular myocardium with occlusion flow < or = 50% of baseline: electrical injury, 25.2 +/- 5.3%; stent, 27.1 +/- 3.6%; balloon, 34.3 +/- 11.6%; P = NS). Furthermore, there were no differences between the animals with thrombosis or balloon occlusion with respect to changes in echocardiographic parameters of left ventricular function, aortic pressure, or heart rate after occlusion. CONCLUSIONS: These data provide evidence that despite equal magnitudes of jeopardized myocardial mass, acute ischemia induced by thrombotic coronary occlusion results in a greater incidence of MVA than does nonthrombotic balloon occlusion. These findings suggest that the process of intracoronary thrombosis itself exerts arrhythmogenic effects above and beyond the impact of ischemia on myocardium induced by coronary occlusion.

Palmitoyl carnitine modifies sodium currents and induces transient inward current in ventricular myocytes.

Long-chain acylcarnitines increase within 2 min in ischemic myocardium in vivo and induce delayed afterdepolarizations (DADs) and complex oscillations of membrane potential in vitro. This study was performed to assess the ionic currents underlying these electrophysiological alterations in isolated rabbit ventricular cells using whole cell voltage-clamp procedures. Palmitoyl carnitine (10 microM, for 6-10 min) elicited a transient inward current (Iti) in the presence of blockade of Ca2+ and K+ channels. The effect of palmitoyl carnitine was reversible after washout (n = 6). The amplitude of Iti was dependent on the amplitude of the preceding depolarization step. Palmitoyl carnitine (10 microM, for > 2 min) also induced another inward current, which was activated spontaneously at potentials between -120 and -20 mV with a linear current-voltage relationship (1.0 +/- 0.1 nA at -80 mV). This current was abolished by replacing extracellular Na+ with tetraethylammonium chloride, indicating that Na+ was the charge carrier. Inactivation of this current was slow (gamma = 885.9 +/- 89.1 ms, n = 12) or incomplete, indicating the appearance of a slow-inactivating Na+ inward current [INa(s)]. Palmitoyl carnitine always induced INa(s) before the appearance of Iti. Intracellular ethylene glycol-bis(beta-amino-ethyl ether)-N,N,N',N'-tetraacetic acid (10 mM) abolished Iti but did not suppress INa(s) (n = 4), indicating that INa(s) was not activated by intracellular Ca2+ (Cai2+). Tetrodotoxin (10 microM) also decreased the amplitude of INa(s). Thus palmitoyl carnitine induces INa(s), which likely leads to an increase in Na+ influx, thereby eliciting an increase in Cai2+ via the Na(+)-Ca2+ exchanger and leading to the development of Iti, DADs, and triggered activity.

Cellular uncoupling induced by accumulation of long-chain acylcarnitine during ischemia.

Long-chain acylcarnitines (LCACs) increase rapidly within minutes after the onset of ischemia in vivo or hypoxia in vitro and produce a time-dependent reversible reduction in gap junctional conductance in isolated myocyte pairs. The present study was performed to assess whether LCACs contribute to cellular uncoupling in response to ischemia in isolated blood-perfused rabbit papillary muscles by use of simultaneous measurements of transmembrane action potentials, extracellular electrograms, extracellular K+, and tissue LCACs and ATP. LCACs increased threefold in response to 20 minutes of no-flow ischemia from 127 +/- 5 to 397 +/- 113 pmol/mg protein (P < .01), concomitant with the onset of cellular uncoupling, extracellular K+ accumulation, and a marked reduction in conduction velocity and action potential duration. To assess whether inhibition of the accumulation of LCACs modified the electrophysiological alterations during ischemia, muscles were pretreated with either sodium 2-(5-(4-chlorophenyl)-pentyl)-oxirane-2-carboxylate (POCA, 10 mumol/L) or oxfenicine (100 mumol/L), inhibitors of carnitine acyltransferase I. Both POCA and oxfenicine completely prevented the increase in LCACs even with 40 minutes of ischemia (138 +/- 37 and 56 +/- 4 pmol/mg protein, respectively), associated with a marked delay in the onset and progression of cellular uncoupling and ischemic contracture. Although POCA and oxfenicine did not affect either the initial early rise in extracellular K+ or the initial fall in conduction velocity, both agents markedly delayed the secondary rise in extracellular K+ as well as the secondary fall in conduction velocity, independent of the level of tissue ATP. Thus, LCACs accumulate during myocardial ischemia and contribute substantially to the initiation of cell-to-cell uncoupling. Inhibition of carnitine acyltransferase I and prevention of the increase in LCACs markedly delays cellular uncoupling and development of ischemic contracture in response to ischemia.

Subcellular distribution of endogenous long chain acylcarnitines during hypoxia in adult canine myocytes.

OBJECTIVE: Previous studies from our laboratory showed a pronounced increase in the sarcolemmal accumulation of long chain acylcarnitines in isolated neonatal rat myocytes after a prolonged (60 minute) hypoxic interval. Because much shorter intervals of hypoxia are associated with electrophysiological alterations in adult cells, the present study was performed to assess the extent of sarcolemmal accumulation of long chain acylcarnitines during hypoxia in adult canine myocytes. METHODS: Cells were incubated (for 24 hours) with 3H-carnitine and the uniformity of incorporation into carnitine fractions was verified biochemically. Cells were exposed to hypoxia (PO2 < 15 mm Hg) for 10 or 20 minutes in the presence or absence of sodium 2-[5-(4-chlorphenyl)-pentyl]-oxirane-2-carboxylate (POCA; 10 microM), an inhibitor of carnitine acyltransferase I. Cells were processed for electron microscopical autoradiography with a technique to spatially fix endogenous long chain acylcarnitines with selective and complete removal of short chain and free carnitine. Grain distributions were analysed by the maximum likelihood method from digitised micrographs. RESULTS: Total mass of long chain acylcarnitines increased ninefold [42.3(3.3) to 374(42) pmol.mg-1 protein] by 10 minutes of hypoxia and 15-fold [to 632(36) nmol.mg-1 protein] by 20 minutes of hypoxia. Normoxic cells exhibited little long chain acylcarnitines in the sarcolemma, and modest amounts in mitochondria and cytoplasm. In hypoxic cells, content of long chain acylcarnitines in mitochondria and cytoplasm increased by a maximum of twofold. By contrast, long chain acylcarnitines increased 100-fold in the sarcolemma to 4.18 x 10(6) molecules.microns-3 after 10 minutes of hypoxia. The increase in long chain acylcarnitines with hypoxia was completely prevented by pretreatment with POCA. CONCLUSION: Hypoxia in adult ventricular myocytes induces a rapid and preferential increase in endogenous long chain acylcarnitines within the sarcolemma.

Thrombin-induced release of lysophosphatidylcholine from endothelial cells.

Lysophosphatidylcholine (LPC) increases extracellularly during ischemia in vivo in both animals and man as judged by measurements from venous effluents, but more recent studies have shown little or no increase in buffer-perfused, isolated heart preparations. The appearance of LPC in blood and lymph in animals and in venous effluents in man in response to ischemia suggests a vascular site for the production of LPC. The present study was performed to assess whether thrombin could stimulate phospholipase A2 in endothelial cells and whether this would evoke an increase in and release of LPC. Endothelial cells were disassociated from canine aortas by incubating with 0.1% collagenase for 20 min. Cells were plated and allowed to grow to confluence. Measurement of LPC was performed using Bligh and Dyer extraction of lipids, high performance liquid chromatography separation, and quantification of LPC using a recently developed radiometric assay employing [3H]acetic anhydride. Incubation of endothelial cells with thrombin (0.05 unit/ml) resulted in a 2.5-fold increase in LPC to 2.3 +/- 0.1 nmol/mg of protein at 2 min (p < 0.01) and returned to control levels within 20 min. The increase in LPC induced by thrombin exhibited a concentration-dependent response with an ED50 = 0.04 unit/ml. A concentration-dependent increase in LPC was also elicited by stimulation with the peptide portion of the thrombin receptor's tethered ligand SFLLRNPNDKYEPF with an ED50 = 8 microM. The LPC produced was rapidly and completely released into the surrounding media. Hirudin completely blocked the thrombin-induced increase in LPC. Dansylarginine N-(3-ethyl-1,5-pentanediyl)amide (0.1 microM), which rapidly inactivates thrombin's proteolytic activity in situ without impairing binding, or phenyl-prolyl-arginyl-chloromethyl ketone (PPACK, 5 nM), which inactivates thrombin due to chemical alteration of the proteolytic site, each prevented the increase in LPC in response to thrombin. Stimulation of protein kinase C with phorbol 12-myristate-13-acetate (PMA, 1 microM) enhanced the response to thrombin. In contrast, staurosporine (100 nM), H7 (15 microM), or chronic treatment with PMA for 20 h to down-regulate protein kinase C completely prevented the increase in LPC in response to thrombin. Thus, thrombin stimulation of endothelial cells in vivo during ischemia may be a primary mechanism contributing to the marked increase in LPC extracellularly during ischemia.

Recent insights pertaining to sarcolemmal phospholipid alterations underlying arrhythmogenesis in the ischemic heart.

Myocardial ischemia in vivo is associated with dramatic electrophysiologic alterations that occur within minutes of cessation of coronary flow and are rapidly reversible with reperfusion. This suggests that subtle and reversible biochemical alterations within or near the sarcolemma may contribute to the electrophysiologic derangements. Our studies have concentrated on two amphipathic metabolites, long-chain acylcarnitines and lysophosphatidylcholine (LPC), which have been shown to increase rapidly in ischemic tissue in vivo and to elicit electrophysiologic derangements in normoxic tissue in vitro. Incorporation of these amphiphiles into the sarcolemma at concentrations of 1 to 2 mole%, elicits profound electrophysiologic derangements analogous to those observed in ischemic myocardium in vivo. The pathophysiological effects of the accumulation of these amphiphiles are thought to be mediated by alterations in the biophysical properties of the sarcolemmal membrane, although there is a possibility of a direct effect upon ion channels. Inhibition of carnitine acyltransferase I (CAT-I) in the ischemic cat heart was found to prevent the increase in long-chain acylcarnitines and LPC and to significantly reduce the incidence of malignant arrhythmias including ventricular tachycardia and fibrillation. This review focuses on the electrophysiologic derangements that are observed during early ischemia and presents data supporting the concept that accumulation of these amphiphiles within the sarcolemma contributes to these changes. The potential contribution of these amphiphiles to the increases in extracellular potassium and intracellular calcium are examined. Finally, recent data pertaining to the accumulation of long-chain acylcarnitines on cell-to-cell uncoupling are presented. In addition to the events reviewed here, there are many other alterations that occur during early myocardial ischemia, but the results from multiple studies over the past two decades indicate that the accumulation of these amphiphiles contributes importantly to arrhythmogenesis and that development of specific inhibitors of CAT-I or phospholipase A2 may be a promising therapeutic strategy to attenuate the incidence of lethal arrhythmias associated with ischemic heart disease in man.

Inhibition of gap junctional conductance by long-chain acylcarnitines and their preferential accumulation in junctional sarcolemma during hypoxia.

Electrophysiological and biochemical sequelae of myocardial ischemia occur within minutes of the onset of myocardial ischemia in vivo. Both conduction delay and conduction block occur rapidly within the same time interval as the accumulation of long-chain acylcarnitines. In the present study, double whole-cell voltage-clamp procedures were used to assess the influence of long-chain acylcarnitines on gap junctional conductance in isolated pairs of canine ventricular myocytes. Long-chain acylcarnitine (5 microM) decreased gap junctional conductance from 153 to 48 nS in a time-dependent and reversible manner. Although the amplitude of junctional current was reduced by 68%, the current continued to demonstrate a linear current-voltage relation. The extent of endogenous accumulation of long-chain acylcarnitines in junctional regions of the sarcolemma was assessed in isolated myocytes in which endogenous free, short-chain, and long-chain acylcarnitine pools had been equilibrated with [3H]carnitine. Under normoxic conditions, long-chain acylcarnitines were not detectable in junctional sarcolemma of myocytes as assessed using electron microscopic autoradiography. Exposure of myocytes to hypoxia (PO2, < 15 mm Hg) for 10 minutes resulted in the preferential accumulation of endogenous long-chain acylcarnitines in junctional sarcolemma (173 +/- 5 x 10(5) molecules/microns 3), a concentration that was sevenfold greater than that found in nonjunctional sarcolemma. Therefore, endogenous long-chain acylcarnitines accumulate preferentially in junctional regions of the sarcolemma during short intervals of hypoxia. Exogenously supplied long-chain acylcarnitines can markedly decrease cellular coupling in a reversible manner, suggesting that this amphiphile may contribute to the marked slowing in conduction velocity in the ischemic heart in vivo, not only by suppressing the rapid Na+ inward current directly, as has been shown previously, but also by decreasing cellular coupling.

Dissociation between cellular K+ loss, reduction in repolarization time, and tissue ATP levels during myocardial hypoxia and ischemia.

The mechanisms underlying the marked increase in [K+]o in response to ischemia are not fully understood. Accordingly, the present study was performed to assess the contribution of ATP-regulated K+ channels by using simultaneous measurements of cellular K+ efflux, [K+]o, transmembrane action potentials, and tissue ATP, ADP, phosphocreatine, and creatine content in a unique isolated, blood-perfused papillary muscle preparation during hypoxia compared with ischemia. During 15 minutes of hypoxic perfusion (PO2, 6.1 +/- 0.9 mm Hg) with normal [K+]o of 4.1 +/- 0.1 mM, action potential duration (APD) was not altered even though tissue ATP levels decreased markedly from 33.5 +/- 1.8 to 14.7 +/- 2.0 nmol.mg protein-1 (p < 0.01). Net cellular K+ efflux, based on measured differences of [K+] between the venous effluent and the perfusate, was 13.23 +/- 0.79 mumol.g wet wt-1 during hypoxia. In contrast, after 15 minutes of zero-flow ischemia, APD at 80% of repolarization (APD80) decreased by 47% from 171 +/- 5 to 92 +/- 5 msec (p < 0.01), but integrated net cellular K+ efflux over 15 minutes of ischemia was 8.4-fold less (1.57 +/- 0.13 mumol.g wet wt-1) than during hypoxia. Tissue ATP levels, however, decreased by only 35.2% to 21.7 +/- 2.1 nmol.mg protein-1, which was significantly less than that induced by 15 minutes of hypoxia. Perfusion with hypoxic blood containing high [K+]o of 10.3 +/- 0.3 mM resulted in APD shortening similar to that observed during ischemia. Cellular K+ loss, however, was inhibited markedly by high [K+]o perfusion (only 4.51 +/- 0.28 mumol.g wet wt-1). Pretreatment with glibenclamide (5 microM), a drug that has been reported to inhibit ATP-regulated K+ channels and accelerate glycolysis in normoxic tissue, partially inhibited cellular K+ efflux during hypoxic perfusion with normal [K+]o (7.35 +/- 0.71 versus 13.23 +/- 0.79 mumol.g wet wt-1, p < 0.01) but had no significant influence on repolarization time or tissue ATP levels. Although glibenclamide partially prevented action potential shortening induced by hypoxic perfusion in the presence of elevated [K+]o, the proportion of cellular K+ efflux reduced by glibenclamide was less (23%) than that observed with glibenclamide in hypoxic perfusion with normal [K+]o (44%).(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphatidic acid stimulates inositol 1,4,5-trisphosphate production in adult cardiac myocytes.

The cellular content of phosphatidic acid can increase in response to several agonists either by phosphorylation of diacylglycerol after phospholipase C-catalyzed hydrolysis of phospholipids or directly through activation of phospholipase D. Although previous findings indicated that the generation of phosphatidic acid was exclusively a means of regulation of the cellular concentration of diacylglycerol, more recent studies have indicated that phosphatidic acid may also directly regulate several cellular functions. Accordingly, the present study was performed to assess whether phosphatidic acid could stimulate cardiac phospholipase C in intact adult rabbit ventricular myocytes. The mass of inositol 1,4,5-trisphosphate [Ins (1,4,5)P3] was determined by a specific and sensitive binding protein assay and by direct mass measurement using anion exchange chromatography for separation of selected inositol phosphates and gas chromatography and mass spectrometry for quantification of inositol monophosphate (IP1), inositol bisphosphate (IP2), inositol trisphosphate (IP3), and inositol tetrakisphosphate (IP4). Phosphatidic acid (10(-9)-10(-6) M) elicited a rapid concentration-dependent increase in Ins (1,4,5)P3 accumulation, with the peak fourfold to fivefold increase at 30 seconds of stimulation; the concentration required for 50% of maximal stimulation was 4.4 x 10(-8) M. The time course of individual inositol phosphates indicated a successive increase in the mass of IP3, IP4, IP2, and IP1 in response to stimulation with phosphatidic acid. The production of Ins (1,4,5)P3 in response to phosphatidic acid was not altered in the absence of extracellular calcium or in the presence of extracellular EGTA (10(-3) M). Thus, these findings indicate that phosphatidic acid is a potent activator of inositol phosphate production in adult ventricular myocytes.(ABSTRACT TRUNCATED AT 250 WORDS)