Oxidative damage in selenium deficient hearts on perfusion with adriamycin: protective role of glutathione peroxidase system

The protective effects of the glutathione peroxidase system against functional damage induced by perfusion of isolated hearts with adriamycin, an anthracycline antibiotic, were studied. We used selenium deficient rats, in which cardiac glutathione peroxidase activity was only 3% of control rats. Both contractile tension and coronary flow decreased during perfusion with the antibiotic. The degree of decline was significantly greater in the selenium deficient hearts than in the control hearts. The increase in malondialdehyde, a product of lipid peroxidation, induced by adriamycin perfusion was more evident in selenium deficient hearts, though the level of reduced glutathione was well maintained. Isolated mitochondrial function also decreased after aerobic adriamycin perfusion and the decrease was greater in selenium deficient rats. These observations indirectly suggest that the decrease in cardiac function induced by adriamycin is protected by the glutathione peroxidase system and that the decrease may be due, at least in part, to damage to the mitochondria caused by oxygen radicals generated by adriamycin.     

Rescue by coenzyme Q10 from electrocardiographic abnormalities caused by the toxicity of adriamycin in the rat.

The administration of adriamycin to rates increased (P less than 0.01) the interval, measured in msec, of the electrocardiographic QRS traces in rats, and the magnitude of the increase was ca. 50%. The administration of coenzyme Q10 to such adriamycin-treated rats allowed "rescue" or restoration of a normal QRS complex after 7 days of administration of coenzyme Q10. The QRS complex then remained normalized during the subsequent period of 21-30 days, by which time the cumulative dose of adriamycin had reached 24 mg/kg. Also, the QRS interval was lower (P less than 0.01) on day 33 than it was for rats treated to the same day with adriamycin alone. Coenzyme Q10 offers promise of rescue from at least some of the cardiotoxicity occurring in adriamycin-treated cancer patients, probably by a similar mechanism to that of the clinical rescue from toxicity of methotrexate by a cofactor of folic acid (citrovorum factor).     

Effect of metoprolol on activity of β-adrenoceptor coupled to guanine nucleotide binding regulatory proteins in adriamycin-induced cardiotoxicity

Summary Prevention of cardiotoxicity without interfering with the therapeutic efficacy of adriamycin is a very crucial question. We have investigated the activity of -adrenoceptor coupled to guanine nucleotide binding regulatory proteins (G-proteins) and Ca²⁺-ATPase activity in experimental adriamycin-induced cardiotoxicity and the influence of metoprolol treatment on these variables. Adriamycin was administered to rats intravenously as a single dose of 6 mg/kg, and metoprolol was continuously given by means of implanted osmotic pumps. -adrenoceptor characteristics were measured by radioligand-binding experiments and by basal and stimulated adenylyl cyclase activity. Northern blot and dot blot analysis was used to quantify G-protein mRNA. It was shown that adriamycin did not induce any change in the total -adrenoceptor density, nor did the high affinity agonist binding to -adrenoceptor change. Adriamycin did not induce any alteration in the amount of mRNA encoding for stimulatory (Gs) or inhibitory (Gi) G-proteins. Also, basal and stimulated adenylyl cyclase activities were identical in the different experimental groups. In contrast, the Ca²⁺-ATPase was shown to increase in adriamycin-treated rats compared to control rats (45 ± 3.8 versus 23 ± 1.2 mol Pi/mg/h, P < .01). Metoprolol was shown to normalize this increase (29 ± 2.1 mol Pi/mg/h). Thus, it may be concluded that in experimental adriamycin-induced cardiotoxicity, despite Ca²⁺-overloading, the -adrenoceptor-G protein-adenylyl cyclase system remains intact. Metoprolol seems to prevent Ca²⁺-overloading independently of the -adrenoceptors studied here.     

Ischemia and reperfusion injury in isolated rat heart: Effect of reperfusion duration on xanthine oxidase,lipid peroxidation,and enzyme antioxidant systems in myocardium

Summary The aim of this work was to assess the catalytic activity of xanthine oxidase, the level of lipid peroxides and enzymic antioxidant systems in isolated rat heart muscle subjected to a globally partial ischemia followed by varying durations of reperfusion.After 40 min of globally partial ischemia (residual perfusion flow rate: 0.1 ml/min), four different durations of reperfusion were investigated (0, 20, 40, and 60 min). After each experimental ischemia/reperfusion sequence, the heart was frozen in liquid nitrogen. Lipid peroxides were assayed in the cardiac homogenate and the catalytic of xanthine oxidase and enzymic antioxidant systems (glutathione peroxidase, superoxide dismutase and catalase) were determined in the centrifuged supernatant. In the different experimental protocols studied in this work, there was no significant increase in the activity of cardiac xanthine oxidase or in the level of lipid peroxides when compared to the non reperfused or to the continuously perfused hearts. Indeed, enzymic antioxidant systems were also not significantly modified in the different periods of reperfusion when compared to control hearts (continuously perfused hearts).These results suggest that xanthine oxidase is apparently not a major source of free radicals in the course of an ischemia-reperfusion sequence in heart muscle, in particular, if we consider the early phases of reperfusion. The process of lipid peroxidation, assessed by assaying thiobarbituric acid reactants, is not a predominant phenomenon of reperfusion-induced injury, at least in the experimental model used here. However, enzymic antioxidant systems investigated in this study do not seem modified. This could mean that the small quantity of oxygen free radicals produced does not overwhelm the enzymic antioxidant systems of myocardium which is in agreement with peroxidatized lipid results.Abbreviations index DMPO dimethyl pyrroline-N-oxide - EPR electron paramagnetic resonance - GPx glutathione peroxidase - LDH lactate dehydrogenase - LP lipid peroxides - OFR oxygen free radicals - SOD superoxide dismutase - TBAR thiobarbituric acid reactants - XD xanthine dehydrogenase - XO xanthine oxidase     

Mitochondrial calcium in hearts subjected to lipid peroxidation with contracture development

Summary In the present study we have investigated isolated rat hearts perfused with oxygen radicals generated by xanthine oxidase and hypoxanthine. The influence of verapamil (1 mg·l^–1) pretreatment on oxygen radical-induced contracture development and decrease in contractility was examined. In addition, we have measured mitochondrial calcium and magnesium levels in control hearts and hearts perfused with oxygen radicals with and without addition of superoxide dismutase (SOD) and catalase. The presence of oxygen radical-induced lipid peroxidation was confirmed by the increased level of conjugated diens in lipid extracts from oxygen radical-perfused hearts. Verapamil prevented contracture development in hearts perfused with oxygen radicals. Diastolic pressure measured with a left ventricle balloon was at the end of the experiments, 18±3 mm Hg (mean±SEM) with verapamil and 66±9 mm Hg without (p<0.001). Perfusion with oxygen radicals resulted in a reduction in mitochondrial calcium from 14.63±0.93 to 8.26±0.61 nmol·mg^–1 (p<0.001) which was partly reversed by superoxide dismutase and catalase. Mitochondrial magnesium levels were unchanged in all groups.     

Age-dependent production of mitochondrial hydrogen peroxide,lipid peroxides and fluorescent pigments in the rat heart

Summary Mitochondria were prepared from hearts of 3-, 14-, 18-, and 24-month-old male Wistar rats. Respiratory control ratio (RCR) values did not change with age in the glutamate or succinate-induced respiration except at 24 months in which RCR values significantly increased with both the substrates. Using still glutamate or succinate as substrates the production of H₂O₂ was measured in the presence of antimycin. A 70% and 25% increase in H₂O₂ formation was observed at 14 and 18 months of age, respectively, in comparison to the youngest group. Only in the presence of succinate was a 25% elevation in H₂O₂ found at 24 months of age. These observations parallel with the decrease of the ratio between tissue levels of reduced and oxidized glutathione that was observed at 14 and 18 months of age. The concentration of myocardial malondialdehyde, a secondary product of lipid peroxidation, remained the same at all ages measured, most probably because it is readly metabolized in vivo. On the contrary the myocardial level of lipofuscin, which is not degraded by the cell, progressively increased beginning from 18 months of age.     

Functional significance of ventricular dilatation

Summary On the basis of theoretical considerations and experimental data this study deals with the functional consequences of structural dilatation, particularly in view of Linzbach's concept of chronic heart failure (34–38). After a short review of the literature, a theoretical analysis of the relationship between stroke volume and ventricular inner radius is presented assuming a thick-walled sphere. Presupposing constant contractility, end-diastolic sarcomere length, end-diastolic wall thickness and end-systolic pressure, only a considerable increase of ventricular radius could be the direct cause of ventricular pumping failure — despite increasing wall stress and reduced ejection fraction. Impaired contractility, as well as insufficient hypertrophy and increased systemic pressure, would intensify the adverse consequences of ventricular enlargement to a predictable extent. Thus, hemodynamic and energetic consequences of dilatation, although mutually interacting, should in principle be distinguished. Despite considerable simplifications involved in model calculations, the relative significance of contractility, ventricular size, wall thickness, and extracardiac factors (mechanical overload; neuroendocrine reactions) can be estimated in various animal models with congestive failure. Hence, this theoretical and experimental approach permits the modification and deepening of previous concepts of structural dilatation and also has implications for interpreting the effects of therapeutical interventions.Supported by the Deutsche Forschungsgemeinschaft (Ja 172/14-1)Dedicated to Dr. Erwin Riesch, Honorary Senator of the University of Tübingen, on the occasion of his 80th birthday     

Lidocaine improves survival rate in diabetic rats submitted to acute left coronary artery ligation

Summary This study was designed to evaluate whether the decreased early survival rate of diabetic rats submitted to acute experimental myocardial infarction can be improved by pretreatment with lidocaine. Male Wistar rats (±210g) were rendered diabetic with i.v. injection of streptozotocin (50 mg/kg), and only those presenting 1 week later a tail-blood glucose value between 250–400 mg/dl were retained in the protocol. Eleven weeks after induction of diabetes, a bolus of lidocaine (2mg/kg) was administered i.v. about 6 min prior to ligation of the left coronary artery under ether anesthesia in control (n=54) and diabetic (n=48) rats; similar studies were conducted in 53 control and 55 diabetic rats without lidocaine pretreatment. Adequate occlusion was confirmed by an elevation of plasma CK-MB levels 4h later or by a toluidine blue injection technique in rats which died earlier. Rats were followed over 48h and comparison in the survival rate in each group established with the Fisher's exact test. Early survival rate (measured after 20 min) was significantly decreased in diabetic rats (27% vs 45%; p=0.04). This was greatly improved by lidocaine pretreatment in diabetic (60% vs 27%; p=0.0013), but not in control animals (50% vs 45%; p=0.384). Furthermore, the beneficial effect of prophylactic lidocaine observed early after coronary ligation in diabetic rats was maintained throughout the period of observation (48h). These data suggest that the prophylactic use of lidocaine is able to reverse the increased incidence of sudden death following experimental myocardial infarction in chronically diabetic rats.     

Superoxide dismutase decreases early reperfusion release of conjugated dienes following regional canine ischemia

Summary Oxygen radical-induced myocardial lipid peroxidation may cause injury during regional ischemia and reperfusion. However, in vivo detection of lipid peroxidation is difficult. Since conjugated dienes are lipid peroxidation products of unsaturated fatty acids, we evaluated the potential value of detection of these double-bonded fatty acids as a marker of oxygen radical injury. In seven untreated and five superoxide dismutase-treated anesthetized dogs exposed to 90 min of coronary occlusion and subsequent reperfusion, coronary sinus plasma draining the ischemic and reperfused region was assayed for dienes. Lipids were extracted and diene optical density measured at 233 nm wavelength. Superoxide dismutase (5 mg/kg, total dose) was infused into the left atrium during ischemia and the first 30 min of reperfusion. Coronary sinus diene optical density increased in untreated animals at 5 and 10 min of reperfusion (reperfusion optical density (x±SEM): 5 min=1.49±0.20 absorbance units, 10 min=1.36±0.06; both p<0.05 vs preocclusion optical density=1.10±0.05 and 25 min reperfusion=1.20±0.07). No increase in diene optical density occurred in superoxide dismutase-treated dogs. Myocardial lipid peroxidation products, as conjugated dienes, increased in coronary sinus plasma during early reperfusion and this increase was prevented by superoxide dismutase infusion.     

Capillary neoformation in the rat heart-stereological studies on papillary muscles in hypertrophy and physiologic growth

Summary Stereological investigations on myocardial capillaries provided evidence that the common estimator of capillarity, the capillary density (i.c., number of capillary profiles per unit transverse sectional area), underestimates the true capillary supply since the capillary axes are not oriented perfectly in parallel to the myofiber axes. Recently, we studied the true capillarity, i.c., the length density of capillaries (L_v=capillary length per capillary volume), in some experimental models of cardiac hypertrophy which have been published elsewhere. It has been shown that L_v decreases in renovascular hypertension, but is maintained in physical exercise and after chronic thyroxin application. However, the growth pattern of capillaries in hypertrophic hearts has not yet been analyzed. In the present paper it is demonstrated that important information on the capillary network can be derived from the two-dimensional capillary-to-fiber ratios (2D CFR: capillary profiles per myofiber profiles in transverse sections) and from the three-dimensional capillary-to-fiber ratios (3D CFR: capillary length per unit myofiber length). Increase in both suggests neoformation of additional capillary branches in parallel connection. Retrospective analysis of the quantitative data indicates that in hypertrophy induced by physical exercise or by chronic thyroxin application capillary neoformation in parallel connection counterbalances increase of oxygen diffusion distance due to myofiber enlargement. In renovascular hypertension, capillary neoformation in parallel connection does not occur. Studies on normal growth indicated both a slight decrease of L_v of capillaries, as well as a continuous neoformation of additional capillary branches.     

Effects of captopril on left ventricular structure and function in SHR with established hypertension

Summary While antihypertensive therapy is considered to be an important clinical intervention in hypertensive patients, its effects on cardiac structure and function have not been intensely evaluated. In this study we tested the hypotheses that lowering blood pressure (BP) with the angiotensin I-converting enzyme inhibitor captopril, would: 1) normalize left ventricular mass and increase the cardiocyte mitochondria/myofibrils volume (V_mito/V_myo) ratio; and 2) not compromise peak ventricular performance. We treated 16-week-old SHR and WKY with captopril (40–80 mg/kg) and hydrochlorothiazide (500 mg/l) via their drinking water. After six weeks of treatment peak cardiac performance was measured during rapid volume overload. Tissue samples from the left ventricular wall were analyzed by electron microscopy and stereology. Captopril lowered BP in SHR and WKY but had no affect on the left ventricular/body weight ratio. The only intracellular change in treated SHR was an increase in sarcoplasmic volume density. Treated WKY exhibited decreased midmyocardial mitochondrial volume density. At peak cardiac output, acceleration of flow and cardiac index were not affected by treatment. Stroke work at peak cardiac output was decreased in the treated groups due to a decrease in mean arterial pressure. In addition, captopril treatment resulted in a shift of the cardiac output (CO)-left ventricular end diastolic pressure (LVEDP) curves, such that LVEDP at peak cardiac output was approximately 50% less in the treated groups compared to their respective control groups. Although captopril was efficacious in lowering BP, it is suggested that lowering BP with this agent does not, at least within six weeks, lead to a reversal of hypertrophy or to a significant alteration in the volume densities of myofibrils and mitochondria. However, an important effect of this antihypertensive drug which may be of clinical significance, is that it leads to a leftward shift of the CO-LVEDP curve in both hypertensive and normotensive rats.     

Coronary vessel tonus and cardiac resistance to ischemia in chronic adriamycin-induced myocardial damage

The rats receiving a cumulative dose of adriamycin, 15-20 mg/kg exhibited delayed weight gain, ascites, and increased relative liver weight. The animals' isolated hearts perfused at a constant rate in the Krebs' solution in a retrograde fashion showed lowered perfusion pressure, indicating reduced coronary vascular tone. At the constant heart rate, the hearts from the adriamycin-treated animals consumed oxygen less at equally increased pressure. In total ischemia, higher cellular lactate and K+ yield occurred than in controls, the severity of acidosis being nearly similar. Prior to ischemia, the pressure produced by the hearts from adriamycin-treated animals was less but, following reperfusion, it more rapidly became normal than that in controls. The results suggest a compensatory increase in myocardial glycolysis during chronic administration of adriamycin.     

SEM observations on the effect of anthracycline drugs on cultured newborn rat cardiomyocytes

Summary The effect of two anthracyclines-doxorubicin hydrochloride (adriamycin) and 4-epidoxorubicin (epirubicin) and an anthracenedione (novantrone) on the contractibility and surface ultrastructure of newborn rat cardiomyocytes cultered for five days was examined. While the beating rate of the cells was affected only by the anthracyclines, an alteration of the sarcolemma, disruption of the slender processes and swelling of the nuclei and/or the cells was observed following incubation with each of the three drugs for two hours. However, the damage induced by adriamycin was more pronounced than that induced by the other two drugs, when doses extrapolated from those accepted as therapeutic were compared.     

Sex differences in the tolerance of immature rat myocardium to global ischemia

Summary Currently there is considerable interest in the metabolism of the immature myocardium and in particular the mechanisms underlying its greater tolerance to ischemia than that of the adult heart. In order to investigate whether this tolerance is sex-related, we compared the recovery of function in isolated hearts from male and female neonatal rats (three to five days old) following 60 min of normothermic global ischemia and 30 min of reperfusion (n=8 per group). The female hearts exhibited significantly better (p<0.05) recovery of rate (81±5% vs. 65±5%) and the rate-pressure product (73±9% vs. 37±8%), and a tendency towards better recovery of contractile function (left ventricular developed pressure, 89±9% vs. 59±12%; dP/dt, 84±12% vs. 54±13%). This evidence for greater resistance of female hearts to ischemic injury was supported by a delayed onset of contracture (mean time to onset, 29.4±2.7 min vs. 24.9±2.6 min). The loss in left ventricular compliance during ischemia and reperfusion was also smaller in the female hearts (increase in left ventricular end diastolic pressure, 6.5±1.2 mm Hg vs. 13.6±3.8 mm Hg). These results suggest that there may be sex-related differences in the tolerance of immature hearts to ischemia, a factor which should be taken into account in the design and interpretation of experimental studies.     

Adaptation to stress exposure prevents arrhythmogenic and contractural effects of the excess of Ca2+ on the heart by the increased activity of sarcoplasmic reticulum

Summary Adaptation of rats to repcated short-term stress exposure prevents, to a considerable extent, contractural and arrhythmogenic effects of high concentrations of extracellular Ca²⁺ on isolated heart. Increased efficiency of SR Ca²⁺-pump functioning and a significant increase in Ca²⁺ pump resistance to autolysis are proved to play the main role in this effect.     

Hemodynamic changes and renal plasma flow in early heart failure: implications for renin,aldosterone, norepinephrine,atrial natriuretic peptide and prostacyclin

Summary Vasoconstrictory and vasodilatory hormone systems may be important in the regulation of peripheral vascular resistance and renal hemodynamics in the carly phase of heart failure. The activity of the renin-angiotensin-aldosterone system (RAAS), the sympathetic nervous activity, and, as possible counterregulating systems, the activity of prostacyclin and atrial natriuretic peptide (ANP) were studied in 6 conscious dogs during the first 4 days of congestive heart failure in relation to hemodynamic changes and renal plasma flow. Congestive heart failure was induced by rapid right ventricular pacing, which caused a considerable decrease of cardiac output (–38%; p<0.05), oxygen saturation of the mixed venous blood (–13%; p<0.05), and mean arterial pressure (–24 mm Hg; p<0.05) on the 4th day. Mean pulmonary arterial pressure and mean pulmonary capillary wedge pressure increased (+4 mm Hg; p<0.05 and +7 mm Hg, respectively; p<0.05). Renal plasma flow was slightly reduced (N.S.), renal vascular resistance did not change. Peripheral vascular resistance showed a significant increase only on the 1st day. Sympathetic nervous activity was stimulated (from 175±31 pg/ml to 391±100 pg/ml; p<0.05), while plasma renin concentration was significantly suppressed on the 4th day (from 3.3±0.4 ngAI/ml/h to 1.9±0.5 ngAI/ml/h; p<0.05), and plasma aldosterone levels were decreased (from 108±12 pg/ml to 76±12 pg/ml; p<0.05). ANP increased 3-fold (p<0.05) and 6-keto-prostaglandin F1 alpha increased in 4 out of 6 dogs. Since ANP is known to inhibit renin release and aldosteronc production, the suppressed RAAS may be an effect of the highly elevated plasma levels of ANP in the early phase of heart failure. The depressor systems such as ANP and prostacyclin may balance the stimulated sympathetic system, resulting in no change of renal blood flow and renal vascular resistance and preventing a considerable increase of peripheral vascular resistance.Supported by the Deutsche Forschungsgemeinschaft     

Enhanced cardiac angiotensinogen gene expression and angiotensin converting enzyme activity in tachypacing-induced heart failure in rats

Summary The aim of the study was to analyze changes in myocardial angiotensinogen gene expression and myocardial angiotensin converting enzyme activity in slowly progressing low-output failure. In adult, male Wistar rats, acute ventricular tachypacing by 610 to 620 impulses per minute lowered enddiastolic external diameter of the left ventricle by 2.6% (p < 0.01), but did not lower cardiac output or abolish coronary reserve, since left-ventricular subendocardial blood flow of paced rats increased under dipyridamole (2 mg/kg i.v.) by 56% (p < 0.01). Systemic neuroendocrine activation and ventricular dilation without enlargement of ventricular mass developed subsequent to chronic tachypacing, but left-ventricular diameter during pacing never exceeded the value of sham rats on sinus rhythm. After 2 weeks, cardiac output was lowered by 14% (p < 0.001), cardiopulmonary blood volume was elevated by 30% (p < 0.001), and angiotensinogen mRNA and angiotensin converting enzyme activity in ventricular myocardium were doubled. We conclude that conditions for an enhanced intracardiac angiotensin II-formation developed in tachypacing-induced heart failure, but that enhanced . systolic wall stress or myocardial ischemia are not required for this activation of the local cardiac reninangiotensin system.     

1,2-Diacylglycerol content in myocardium from spontaneously hypertensive rats during the development of hypertension

Summary 1,2-Diacylglycerol (DAG) has been considered to play an important role as an activator of protein kinase C in the signal transduction of inositol phospholipid metabolism. To examine the relation of 1,2-DAG in heart tissues to cardiac hypertrophy associated with hypertension, we measured the amount of 1,2-DAG in spontaneously hypertensive rat (SHR) hearts at 4,10 and 20 weeks of age, and in age-matched normotensive Wistar-Kyoto (WKY) rat hearts using thin-layer chromatography with flame ionization detection (TLC-FID). Significant cardiac hypertrophy was found in 4-week-old SHR, while SHR did not yet have significant hypertension. Major phospholipids such as phosphatidylcholine and phosphatidylethanolamine increased from 4 to 20 weeks in the myocardium, but there was no difference between the two strains. The cholesterol levels of 4- and 20-week-old SHR were significantly higher than WKY rats. The 1,2-DAG contents of SHR hearts were significantly higher than WKY rats at 4 weeks. An increase in the RNA contents of SHR hearts were significantly higher than WKY rats at 4 weeks. An increase in the RNA content was also observed in 4-week-old SHR hearts. However, analysis of the fatty acid composition of 1,2-DAG revealed no difference between the two strains. However, there was no significant difference in the 1,2-DAG content or in its fatty acid composition between SHR and WKY rat hearts at 10 and 20 weeks of age. It is suggested that an increase in the 1,2-DAG content of SHR hearts during the early stages appears related to the initiation of cardiac hypertrophy in SHR hearts before developed hypertension.     

Ouabain arrhythmogenicity in hypertrophied right and left ventricle of the rat

Summary Susceptibility to the arrhythmogenic action of ouabain was tested in rats with right ventricular hypertrophy, due to experimental chronic hypoxic pulmonary hypertension, and in spontaneously hypertensive (SH) rats with left ventricular hypertrophy. As parameters of arrhythmogenicity the time-duration of infusion of a solution of ouabain (1 g/100 ml), at a rate of 0.7 mg/kg per minute, was measured until the appearance on the ECG of the first premature ventricular contraction, ventricular tachycardia and cardiac arrest. All three effects of digitalis toxicity (premature ventricular contraction, ventricular tachycardia and cardiac arrest) appeared significantly earlier both in rats with right ventricular hypertrophy, due to chronic experimental hypoxic pulmonary hypertension, and in SH rats with hypertrophy of the left ventricle, as compared to the infusion time of the same solution of ouabain needed to elicit the mentioned toxic effects in control rats without ventricular hypertrophy.This work was supported by a grant from the Serbian Research Foundation