Altered ionic calcium and cell motion in ventricular myocytes after cutaneous thermal injury.

Cutaneous thermal injury resulting in burns covering approximately 45% of the total body surface area initiates metabolic alterations which contribute to subsequent myocardial dysfunction. Alterations in calcium homeostasis have been proposed as one mechanism by which burn injury alters organ function. This study used fura-2 and time-resolved single cell fluorescence microscopy to examine stress-related alterations in intracellular calcium in isolated adult rat cardiac myocytes. Ventricular myocytes were isolated from rats given a full-thickness scald burn comprising 43% of the total body surface area and fluid resuscitated with lactated Ringer's by the Parkland formula; control animals were included for comparison. Burn trauma caused a significant increase in cardiac myocyte maximal (peak systolic) and minimal (diastolic) mean cytosolic free calcium concentration ([Ca2+]i) transient ratios when compared to [Ca2+]i transient ratios measured in control rats. Isoproterenol application altered the time course of the [Ca2+]i transients of normal myocytes but this response was not observed in myocytes from the thermally injured rats. In addition, isoproterenol application to normal myocytes produced a significant increase in the amplitude of cell edge motion (+50%) compared to the cell edge motion measured in myocytes without isoproterenol stimulation; however, this cell motion response did not occur after isoproterenol application to myocytes from thermally injured rats. Caffeine application increased the maximal and minimal [Ca2+]i transient ratios of all myocytes, regardless of a burn injury, and the time course of the [Ca2+]i transients from the two groups appeared similar in the presence of caffeine as the myocytes progressed to contracture. Our data suggest that burn-mediated alterations in calcium homeostasis contribute, in part, to the cardiac contractile dysfunction which occurs after burn injury.

Effects of different oxidative insults on intermediary metabolism in isolated perfused rat hearts.

13C and 31P NMR were used to evaluate exogenous substrate utilization and endogenous phosphate metabolites in perfused rat hearts exposed to tert-butylhydroperoxide (tert-BOOH) and hydrogen peroxide (H2O2). Both reagents caused a reduction in developed pressure compared to controls and, in agreement with previous 31P NMR data, had different effects on intracellular high-energy phosphates and glycolysis. 13C Isotopomer analysis of tissue extracts showed that H2O2 and tert-BOOH also had significantly different effects on substrate utilization by the citric acid cycle. The contribution of exogenous lactate and glucose to acetyl-CoA was 43% in controls and increased to over 80% in the presence of either oxidant. With tert-BOOH, exogenous glucose and lactate were both significant contributors to acetyl-CoA (44 +/- 2 and 41 +/- 3%). However, with H2O2, exogenous lactate supplied a much higher fraction of acetyl-CoA (72 +/- 2%) than glucose (9 +/- 1%). Also, when [2-(13)C] glucose was supplied, accumulation of [2-(13)C] and [5-(13)C] fructose 1,6-bisphosphate was observed in the presence of H2O2, indicating inhibition of glyceraldehyde-3-phosphate dehydrogenase. These results indicate that despite this glycolytic inhibition, H2O2 increased the utilization of pyruvate precursors when lactate was present as an alternative carbohydrate substrate.

The role of toxic oxygen metabolites in a young model of thermal injury.

Production of oxygen-free radicals has been proposed as one pathophysiologic mechanism for postburn cardiac contractile dysfunction in adults. To examine this hypothesis in young subjects, we studied the cardiac effects of polyethylene glycol-superoxide dismutase (PEG-SOD) and PEG-catalase (PEG-CAT), each given as 20 U/g of body weight with fluid resuscitation (Parkland formula), after a third-degree burn constituting 33% of the total body surface area in young (6- to 7-day old) guinea pigs (group 3, n = 12). Fluid-treated burns without scavenger therapy (group 2, n = 15) and sham burn controls (group 1, n = 15) were included. Animals were killed 24 hours postburn, and hearts were studied in vitro (Langendorff). Compared with sham burn controls, fluid-treated burns (group 2) had significant cardiac dysfunction as indicated by a lower peak systolic left ventricular (LV) pressure (LVP: 67 +/- 2 vs. 57 +/- 4 mm Hg, p = 0.01, mean +/- SEM), maximal rate of LV pressure development (+dP/dt max: 1169 +/- 45 vs. 988 +/- 45 mm Hg/second, p = 0.01), and fall (-dP/dt max: 1109 +/- 45 vs. 919 +/- 49 mm Hg/second, p = 0.01). In addition, LV function curves calculated for group 2 were shifted downward and to the right of those calculated for sham burn controls in the direction of contractile depression, p = 0.01. PEG-SOD/PEG-CAT treatment in burns did not significantly improve LVP (60 +/- 5 mm Hg), but scavenger therapy improved +/-dP/dt max values (1112 +/- 74 and 988 +/- 98 mm Hg/second, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of 21-aminosteroids in neonatal rat cardiac myocyte cell cultures exposed to free radicals.

OBJECTIVE: The aim was to test a group of 21-aminosteroids, U74006F, U75412E, and U74500E, known as lazaroids, for their ability to prevent alterations in neonatal rat cardiac myocytes exposed to solutions containing a xanthine oxidase mediated free radical generating system. METHODS: Myocytes were either left untreated (non-treated cultures) or pretreated for 15 min with the drug vehicle or one of the lazaroids. Myocytes were either examined as non-exposed control cultures or exposed to the free radical generating system for 60 min. Measurement of [3H]arachidonate label in a lipid extract of the culture medium and release of lactate dehydrogenase (LDH) into the medium were analysed. RESULTS: Myocytes not treated with lazaroids and vehicle treated myocytes exposed to free radicals showed a significant release of [3H]arachidonate and lactate dehydrogenase (LDH). At a dose 1 x 10(-5) M, all lazaroid treated myocytes showed significantly lower release of [3H]arachidonate measured in the total lipid extract compared to the non-treated or vehicle treated cultures. Release of [3H]arachidonate was significantly lower for the myocytes treated with U74006F and U74500E at 1 x 10(-6) M concentration. Only the U74006F treated myocytes showed protection at 1 x 10(7) M. LDH release was significantly attenuated at a dose of 1 x 10(-5) M for the U75412E treated myocytes and at 1 x 10(-5) and 1 x 10(-6) M for the U74500E treated myocytes compared to the myocytes not pretreated with a lazaroid and exposed to free radicals. CONCLUSIONS: Lazaroids provide protection against the release of [3H]arachidonate and LDH from myocardial cells exposed to free radical mediated injury. U74006F appeared to have the higher efficacy, at equal molar concentrations, in protecting against the release of [3H]arachidonate, whereas U74500E was observed to have the higher potency in inhibiting LDH release.

Effects of oxidant exposure on substrate utilization and high-energy phosphates in isolated rat hearts.

The effects of a xanthine oxidase-mediated free radical-generating system containing purine and iron-loaded transferrin or solutions containing hydrogen peroxide and iron-loaded transferrin on substrate utilization and high-energy phosphates were evaluated by nuclear magnetic resonance (NMR) spectroscopy in isolated perfused rat hearts. Hearts were supplied with lactate, acetate, and glucose, and the contribution of each substrate to acetyl coenzyme A was measured in control hearts and in the presence of a free radical-generating system. Perfused hearts were monitored by 31P NMR, and tissue extracts were analyzed by 13C NMR. Free radicals decreased the phosphocreatine and beta-ATP peak areas and reduced contractile function. Under control conditions, lactate, acetate, and endogenous sources were the major contributors of acetyl coenzyme A units, with only 5% originating from glucose. In the presence of a xanthine oxidase-mediated free radical-generating system, the glucose contribution increased to 54%, while contributions from acetate and endogenous sources were significantly reduced. Both 13C and 31P NMR analyses showed no significant accumulation of glycolytic sugar phosphates, suggesting little inhibition of glyceraldehyde-3-phosphate dehydrogenase. The increased contribution of glucose to the tricarboxylic acid cycle relative to acetate and endogenous sources is consistent with activation of pyruvate dehydrogenase. In contrast, hearts exposed to a hydrogen peroxide-based free radical-generating system showed an increase in lactate utilization, a decrease in acetate utilization, and no change in glucose utilization compared with control hearts. Glycolytic sugar phosphates were found to accumulate, suggesting possible inhibition of glyceraldehyde-3-phosphate. Thus, different radicals or their metabolites may have varying effects on myocardial metabolism.

Exposure to free radicals alters ionic calcium transients in isolated adult rat cardiac myocytes.

Oxygen-derived free radical production has been documented to occur on reperfusion of the ischemic myocardium. Intracellular ionic calcium ([Ca++]i) levels in isolated adult rat cardiac myocytes exposed to free radicals were evaluated using fura-2. The effect of different time periods of free radical exposure on altering [Ca++]i was examined. Myocytes were either exposed to the free radical generating system continuously or exposed for 5 or 10 minutes and then returned to the HEPES buffer. Myocytes maintained in HEPES buffer or the HEPES buffer containing purine and iron-loaded transferrin continued to stimulate, exhibited relatively uniform 340/380 nm ratios and maintained a rod shape. Continuous exposure to free radicals resulted in a significant increase in [Ca++]i. Myocytes became unresponsive to stimulation at 31 +/- 7 (SE) minutes and eventually exhibited contracture. Exposure to the free radical generating system for 10 minutes resulted in a response similar to continuous exposure. Myocytes exposed to the generating system for 5 minutes exhibited regular calcium transients for 55 +/- 5 minutes. Thus, even a brief period of free radical exposure alters calcium flux and may induce subsequent myocardial damage.

Free radicals alter ionic calcium levels and membrane phospholipids in cultured rat ventricular myocytes.

Oxygen-derived free radicals have been implicated in damage to membrane phospholipids leading to alterations in membrane function. The purpose of this study was to investigate alterations in intracellular ionic calcium (Ca2+) levels and Ca2+ transients, cellular morphology, conjugated diene levels, arachidonate release, and lactate dehydrogenase release resulting from the exposure of cultured neonatal rat ventricular myocytes to a xanthine oxidase catalyzed free radical generating system capable of producing superoxide and hydroxyl radicals. The ability of alpha-tocopherol to prevent alterations due to free radical exposure was investigated. For measurements of Ca2+, myocytes grown on coverslips for 3-4 days were loaded with fura-2/AM and studied by microspectrofluorometry. Control myocytes superfused with a physiological buffer or buffer containing purine and iron-loaded transferrin exhibited Ca2+ transients associated with spontaneous contractions. For control, buffer perfused myocytes (n = 4), the fura-2 340/380 ratios were 0.5 +/- 0.1 (mean +/- S.E.) and 1.6 +/- 0.03 at the minimum and maximum, respectively, of the Ca2+ transient, after 1 h of perfusion. Exposure to the free radical generating solution (n = 14) altered intracellular Ca2+. The 340/380 minimum ratio was 639% of the control value after approximately 30-70 mins with cessation of normal Ca2+ transients. Bleb development was associated with increased Ca2+. Myocytes reperfused with control medium continued to exhibit an elevated minimum fura-2 ratio at 687% of control. Myocytes pretreated with 10 microM alpha-tocopherol (n = 13) for 18-24 h and exposed to free radicals did not exhibit increases in intracellular Ca2+, having a minimum 340/380 ratio of 0.5 +/- 0.1 after 60-90 mins, and although myocytes often ceased contracting, they resumed spontaneous Ca2+ transients with control medium reperfusion and also maintained normal structure. Exposure of myocyte cultures to free radical generating solutions resulted in increased levels of conjugated dienes and increased release of [3H]arachidonate and lactate dehydrogenase compared to control values after 1 h. alpha-Tocopherol treatment attenuated the increase in conjugated diene levels, and the release of [3H]arachidonate and lactate dehydrogenase. Thus, free radicals alter intracellular Ca2+, conjugated dienes and membrane structure indicating their ability to induce altered ionic homeostasis in association with myocardial membrane damage. alpha-Tocopherol decreased free radical mediated injury.

Free radical damage in neonatal rat cardiac myocyte cultures: effects of alpha-tocopherol, Trolox, and phytol.

A number of investigations have implicated free radicals in the progression of ischemic/reperfusion injury. alpha-Tocopherol has been found to attenuate alterations due to ischemia and reperfusion in an isolated heart model. The present study was intended to directly examine neonatal rat cardiac ventricular cell cultures exposed to a free radical generating system catalyzed by xanthine oxidase. The effectiveness of alpha-tocopherol in the attenuation of the resultant changes and the mechanism by which the effects of alpha-tocopherol may be exerted were evaluated. Cultures were either nontreated or pretreated for 18 h with 20 microM alpha-tocopherol or the subcomponents of the alpha-tocopherol molecule, phytol and Trolox. Exposure of cell cultures to free radicals resulted in significant increases in lipid peroxidation products, release of both lactate dehydrogenase and 3H-arachidonate, and structural alterations. Pretreatment with alpha-tocopherol showed significant attenuation of the changes associated with exposure to free radicals. Trolox and phytol at equal molar doses were not as effective as alpha-tocopherol in protecting the myocytes against injury. Thus, alpha-tocopherol seems beneficial in its ability to reduce free radical-mediated changes by functioning as a lipophilic antioxidant. Additionally, the intact, native alpha-tocopherol molecule exceeded the protective capabilities of either of its subcomponents.

Tension and electrolyte changes with Na+-K+ pump inhibition in rat papillary muscle.

Myocardial ischemic injury results in altered membrane integrity, energy depletion, and electrolyte shifts leading to accumulation of intracellular Ca. However, analysis of the direct effects of Ca accumulation is complicated by other concomitant cellular changes produced by ischemia. The purpose of this study was to examine the effects of Ca loading in rat papillary muscles produced by Na+-K+ pump inhibition in oxygenated K+-free buffer. Changes in contractile characteristics, high energy phosphate, and elemental concentrations of subcellular compartments were measured. Electron probe X-ray microanalysis was used to assess elemental concentrations in cryosections. After 3 h of Na+-K+ pump inhibition, resting tension (RT) increased to 164% and developed tension (DT) fell to 16.8% of control values. One hour after return to complete buffer, RT and DT partially recovered but remained significantly different from the 180 to 240-min values for the control muscles. Electron probe X-ray microanalysis showed increases in cytoplasmic and mitochondrial Na and Ca and a decrease in K during Na+-K+ pump inhibition. Mitochondrial Ca was greater than 100-fold greater than Ca in control mitochondria. Morphologically, the majority of cells showed ultrastructural damage. The mean ATP level was 20% of control. After 1 h of recovery, the cells appeared more heterogeneous, and the mean mitochondrial Ca decreased, whereas mean cytoplasmic Ca increased. Further statistical analysis showed a bimodal distribution for Na, Ca, K, Mg, and Cl, which coincided with the morphologically mixed population of cells. This suggests that replacement of extracellular K+ was associated with restored electrolyte gradients in some cells and the persistent or further alteration of electrolytes in others. These results suggest that variable Ca accumulation and associated ATP depletion without the compounding effects of ischemia lead to cell injury similar to reperfusion injury reported in ischemic myocardium.

Static contraction increases arachidonic acid levels in gastrocnemius muscles of cats.

Static contraction of hind-limb muscles is well known to increase reflexly cardiovascular function. Recently, blockade of cyclooxygenase activity has been reported to attenuate the reflex pressor response to contraction, a finding which suggests that working skeletal muscle releases arachidonic acid metabolites. Therefore, we measured the effects of static contraction and ischemia on arachidonic acid levels in the gastrocnemius muscles of barbiturate-anesthetized cats treated with indomethacin. Unesterified arachidonic acid levels were measured by high-pressure liquid chromatography. We found that static contraction of freely perfused gastrocnemius muscles increased arachidonic acid levels from 4.4 +/- 1.0 to 10.3 +/- 2.2 nmol/g wet wt (n = 12; P less than 0.005). Likewise, static contraction of gastrocnemius muscles made ischemic for 2 min before the onset of the contraction period increased arachidonic acid levels from 12.6 +/- 2.3 to 21.0 +/- 2.0 nmol/g wet wt (n = 12; P less than 0.01). Lastly, 2 min of ischemia with the gastrocnemius muscles at rest increased arachidonic acid levels from 5.9 +/- 1.1 to 10.5 +/- 3.0 nmol/g wet wt (n = 18; P less than 0.02). We conclude that both static contraction and ischemia increase arachidonic acid levels in working hindlimb muscle.

alpha-Tocopherol attenuates myocardial membrane-related alterations resulting from ischemia and reperfusion.

Myocardial ischemia and reperfusion have been shown to result in damage to the phospholipid components of cardiac myocyte cell membranes as indicated by the tissue accumulation of unesterified fatty acids (UFA). A portion of this damage and subsequent dysfunction may be a consequence of free radical-induced membrane lipid peroxidative events. alpha-Tocopherol, a lipophilic antioxidant, was used in this study as an agent by which the extent of ischemia and reperfusion injury might be decreased. Increasing rat myocardial tissue levels of alpha-tocopherol by 51% was found to attenuate lipid perturbations as determined by the accumulation of tissue UFA in an isolated heart model of global ischemia and reperfusion. Nontreated hearts made ischemic for 25 min with 30 min of reflow had a significantly increased total UFA level of 5,961 +/- 799 nmol/mg protein (P less than 0.05) compared with control perfused hearts containing 3,116 +/- 463 nmol UFA/mg protein and with alpha-tocopherol-treated ischemic and reperfused hearts containing 3,066 +/- 365 nmol UFA/mg protein. Contractile dysfunction, excessive accumulation of tissue calcium, and release of lactate dehydrogenase after ischemia and reperfusion were also reduced, demonstrating protective effects in alpha-tocopherol-treated hearts. Thus alpha-tocopherol proved effective in the attenuation of ischemia and reperfusion damage. These results suggest that reducing lipid alterations may prove beneficial in protecting against membrane damage subsequent to ischemia and reperfusion.

Ryanodine and caffeine prevent ventricular arrhythmias during acute myocardial ischemia and reperfusion in rat heart.

This study investigates the possible role of oscillatory release of calcium from sarcoplasmic reticulum in the genesis of ventricular arrhythmias during acute myocardial ischemia and reperfusion in isolated rat hearts. We used ryanodine and caffeine, which are known to modulate the oscillatory release of calcium from sarcoplasmic reticulum. During 30 minutes of left main coronary artery ligation, all 13 control hearts developed ventricular premature beats (number of beats, 225 +/- 51) and ventricular tachycardia (duration, 123 +/- 21 seconds); five hearts developed ventricular fibrillation. In a separate series of experiments, reperfusion after 15 minutes of coronary artery ligation caused ventricular fibrillation to occur within 15 seconds in all 12 hearts. Ryanodine (10(-9) to 10(-7) M) abolished ventricular arrhythmias during coronary artery ligation and prevented reperfusion ventricular fibrillation. Ryanodine (10(-9), 10(-8), and 10(-7) M) caused 15%, 23%, and 74% decreases in the maximal rate of rise of left ventricular pressure development and 20%, 32%, and 85% decreases in the maximal rate of fall of left ventricular pressure development, respectively, prior to coronary artery ligation. During acute myocardial ischemia, ryanodine 10(-9) M maintained and 10(-8) M impaired left ventricular function; 10(-7) M caused left ventricular failure. Coronary perfusion rate did not increase during ischemia. Antiarrhythmic activity occurred independent of preservation of high energy phosphates, reduction in tissue lactate, or tissue cyclic adenosine monophosphate in the ischemic myocardium. Caffeine 10(2) M decreased the incidence of ventricular arrhythmias during ischemia and upon reperfusion; protection occurred coincident with development of diastolic contracture. Caffeine increased ischemic tissue cyclic adenosine monophosphate content and worsened tissue energy status.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence of direct toxic effects of free radicals on the myocardium.

The hypothesis that oxygen-derived free radicals do indeed play a role in myocardial ischemic and reperfusion injury has received a lot of support. Experimental results have shown that free radical scavengers can protect against certain aspects of myocardial ischemic injury and that on reperfusion the heart approaches a level that is more normal than those hearts not receiving additional scavenging agents. Superoxide dismutase, catalase, glutathione peroxidase, hydroxyl radical scavengers and iron chelators such as desferrioxamine have proven successful in providing an increased level of recovery. These results indicate, as would be expected, that superoxide, hydrogen peroxide and hydroxyl radicals may all, at some point, either contribute to the injury or be important in generating a subsequent radical which causes damage. In addition, solutions capable of generating free radicals have been shown to cause damage to myocardial cells and the vascular endothelium that is similar to the damage observed during myocardial ischemic and reperfusion injury. Alterations in function, structure, flow, and membrane biochemistry have been documented and compared to ischemic injury. The continued investigation of the role of free radicals in ischemic injury is warranted in the hope of further elucidating the mechanisms involved in free radical injury, the sources of their generation, and in defining a treatment that will provide significant protection against this particular aspect of ischemic damage.

Accumulation of arachidonate in triacylglycerols and unesterified fatty acids during ischemia and reflow in the isolated rat heart. Correlation with the loss of contractile function and the development of calcium overload.

Alterations in triacylglycerol and phospholipid metabolism are known to occur during the evolution of myocardial ischemic injury. The purpose of this study was to explore potential relationships between the accumulation of arachidonic acid and other fatty acids, the accumulation of triacylglycerol, and the progression of myocardial injury. Measurements of the fatty acid levels in triacylglycerol, unesterified fatty acids, and calcium content were correlated with myocardial function during ischemia and ischemia with reflow in an isolated perfused rat heart preparation. After 10 minutes of ischemia in this model, myocardial dysfunction was reversible, with recovery of left ventricular +dP/dt to 82.0% +/- 4.8% of control values upon reperfusion. Hearts did not recover with reperfusion after 30 minutes of ischemia and displayed a significant increase in tissue calcium content. A significant, nearly threefold increase in the arachidonic acid content of triacylglycerol was found after 10 minutes of ischemia and continued to increase with longer periods of ischemia and reflow. Other fatty acids also showed increased levels in triacylglycerol. The time course of accumulation of unesterified arachidonic acid paralleled the loss of myocardial function. Levels of free arachidonic acid were (in nanomoles per gram wet weight) 11.1 +/- 2.1 (SEM) for control hearts, 17.3 +/- 1.9 after 10 minutes of ischemia, and 38.4 +/- 2.5 after 30 minutes of ischemia. Increases in other free fatty acids contributed to a significant increase in total free fatty acid accumulation after 30 minutes of ischemia. Thus, the content of arachidonic and other fatty acids in triacylglycerol was found to increase early during ischemia, and a major increase in free arachidonic and other unesterified fatty acids occurred after a longer period of ischemia. These findings are consistent with an initial reincorporation of free fatty acids into triacylglycerol after release from membrane phospholipids, suggesting that membrane fatty acids may be a major source of triacylglycerol that accumulates in ischemic myocardium. In addition, these results suggest that a major increase in free fatty acids during ischemia and ischemia with reflow correlates temporally with the development of severe contractile dysfunction and accumulation of calcium in the heart.

Superoxide dismutase enhances recovery following myocardial ischemia.

Oxygen-derived free radicals, specifically superoxide (O-2) and the hydroxyl radical (OH.), have been implicated as possible mediators in the development of myocardial damage induced by ischemia and reflow. The purpose of this study was to examine the ability of superoxide dismutase (SOD), a O-2 scavenging enzyme, to protect the heart against functional and structural alterations due to ischemia and reflow. An isolated perfused rabbit interventricular septal preparation was used for these experiments. Septa were treated with SOD by adding either 10 or 20 micrograms/ml of the enzyme to the perfusion solution 15 min prior to ischemia and during reflow. Other septa were not treated. Septa were made ischemic for 1 h and reperfused for 1 h. The contractile performance of reperfused septa was found to be significantly improved in SOD-treated septa when compared with nontreated septa. After 60 min of reflow, values for nontreated, 10- and 20-micrograms/ml SOD-treated septa, respectively, were 48.5 +/- 5.2 (SE), 67.4 +/- 4.2, and 82.0 +/- 3.8% of control values for developed tension. The rise in resting tension observed with reflow was significantly decreased. SOD treatment also provided significant protection of myocardial ultrastructure. The percent of myocytes showing normal structure was increased approximately 40%, and the percentages of myocytes showing mild or severe damage were decreased approximately 30 and 15%, respectively, for SOD-treated septa. Vessel structure showed a similar trend. Thus SOD preserves myocardial function and structure in septa reperfused following ischemia. These results support the possibility that oxygen-derived free radicals may be involved in the damage resulting from ischemia and reflow.

Myocardial alterations due to free-radical generation.

Oxygen-derived free radicals have been proposed as general mediators of tissue injury in a variety of disease states. Recent interest has focused on the possibility that free radicals may be involved in ischemic myocardial damage. However, the exact types of damage that result from myocardial exposure to free radicals remains to be established. The purpose of this study was to evaluate the effects of superoxide and hydroxyl radicals on myocardial structure and function in an isolated perfused rabbit interventricular septal preparation. Superoxide was generated by adding purine (2.3 mM) and xanthine oxidase (0.01 U/ml) to the physiological solutions perfusing the septa. Hydroxyl radical generation was catalyzed by the addition of 2.4 microM Fe3+-loaded transferrin to the system. Exposure of normal septa to superoxide-generating solutions resulted in the development of structural alterations in the vascular endothelium including the development of vacuoles. Membranous cellular debris was evident in the extracellular space and within the vessels. Cardiac myocytes showed evidence of mild alterations. Exposure of septa to solutions capable of generating hydroxyl radicals resulted in more extensive and severe damage. Vascular endothelial cells showed evidence of vacuoles or blebs and edema. Severe swelling of mitochondria was evident in cardiac myocytes and vascular endothelial cells. In addition, myocytes often showed blebbing of the basement membrane. Normal septa exposed to superoxide showed no significant decrease in developed tension, whereas hydroxyl radical exposure resulted in a significant decrease in myocardial function.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitative x-ray microanalysis of the elemental composition of individual myocytes in hypoxic rabbit myocardium.

The purpose of this study was to use energy dispersive x-ray microanalysis to test the following hypotheses: (1) that individual myocytes may exhibit important variation in the severity of alterations in intracellular ionic homeostasis in response to hypoxia and (2) that hypoxic myocytes may accumulate certain elements in quantities sufficient to impair organellar function and structure. A rabbit interventricular septal preparation with attached small right ventricular papillary muscles was used to obtain control oxygenated myocardium (six papillary muscles) and myocardium rendered hypoxic for 1 to 1 1/2 hr (n = 8). Myocardium not perfused in vitro was also obtained (n = 4). Microanalysis was performed on freeze-dried thin sections of unfixed papillary muscles. Elemental concentrations were determined by suitable cryostandards of elements of interest. Sarcoplasm and mitochondria of most hypoxic myocytes exhibited significant alterations of diffusible elements, including increases in sodium and chloride and decreases in potassium, phosphorus, and magnesium, without major change in calcium. The most severely altered myocytes showed evidence of calcium overloading manifested by markedly increased levels of mitochondrial calcium and phosphorus associated with formation of electron-dense mitochondrial inclusions. Levels of mitochondrial calcium and phosphorus exceeded those previously found to markedly impair the function and structure of isolated mitochondria. Thus x-ray microanalysis of unfixed cryosections provides direct measurements of subcellular alterations in elemental composition of individual myocytes in injured myocardium and demonstrates that both calcium and phosphorus accumulate in mitochondria of severely injured myocytes in concentrations sufficient to exert deleterious effects on these organelles.

Phenothiazine protection in calcium overload-induced heart failure: a possible role for calmodulin.

The effect of several phenothiazines on the extent of cellular damage resulting from the calcium paradox was examined. Hearts treated with trifluoperazine, a potent calmodulin inhibitor, exhibited less cellular damage than untreated myocardium as reflected by light microscopy, high-energy phosphate content and the loss of protein and creatine phosphokinase into the perfusate. A dose response of this effect revealed a maximal response at about 1 microM trifluoperazine, a concentration which lies well within the range generally attributed to calmodulin inhibition. Several other lines of evidence were also obtained suggesting a possible role for calmodulin in calcium-overload induced necrosis. First, the phenothiazines had little influence on membrane changes believed responsible for altered calcium permeability. Second, trifluoperazine was without major effect unless included in the reperfusion buffer, indicating that the drug is only effective during the phase associated with calcium overload. Finally, less protection was afforded hearts exposed to phenothiazines such as chlorpromazine and promethazine, which are weaker inhibitors of calmodulin, than those treated with the potent inhibitor trifluoperazine. While other interpretations are possible, these studies are consistent with a role for calmodulin in calcium overload-induced heart failure.