Furazolidone-induced Cardiomyopathy Research Articles

Calreticulin is localized at mitochondria of rat cardiomyocytes and affected by furazolidone.

Calreticulin (CRT) is a calcium-buffering protein which is predominantly located in endoplasmic reticulum. In the previous mitochondria proteome analysis, we accidentally found that CRT may be also localized at myocardial mitochondria and was upregulated in a rat model of furazolidone-induced dilated cardiomyopathy. To our knowledge, there has not yet been any report of its presence in mitochondria of any cell types. The present study aimed to determine whether CRT was located at the mitochondria of rat cardiomyocytes and whether the mitochondrial CRT was affected by furazolidone. Mitochondrial preparations were isolated from primary cultured neonatal rat cardiomyocytes and purified by differential centrifugation. The purity of mitochondria was assessed by the reduction or elimination of the immunoreactivities of markers for cytosol, nucleus, sarcolemma, and endoplasmic reticulum. Western blot analysis demonstrated the presence of CRT in purified mitochondria of rat cardiomyocytes. The distribution of CRT to mitochondria was further confirmed by immuno-electron microscopy, flow cytometry, and laser scanning confocal microscopy (double staining with MitoTracker Red and CRT-Alexa Fluor 488). Western blot analysis also demonstrated that the mitochondrial content of CRT was significantly enhanced by furazolidone treatment by 2.73±0.13 fold (P<0.05) in rat cardiomyocytes, which was verified by immuno-electron microscopy. In summary, the present results suggest that CRT is localized at mitochondria of rat cardiomyocytes and such localization is affected by furazolidone.

Chronic administration of cardiovascular drugs: altered energetics and transmembrane signaling.

The effects of clinically used cardioactive agents in furazolidone-induced cardiomyopathy in the turkey poult have been recently reported, and note-worthy differences in cardioprotective efficacy of adrenergic effectors, calcium channel blockers, and cardiac glycosides have been noted in animal and human studies of heart failure. We therefore investigated the effects of chronic oral administration of cardioactive agents on ventricular tissue from normal turkey poults, and we determined whether these agents altered cardiac function, energetics, or transmembrane signaling pathways in a manner that might contribute to the varying degrees of cardioprotection and therapeutic efficacy reported previously. Creatine content was significantly higher in propranolol- and atenolol-treated animals. There was also higher lactate dehydrogenase and creatine kinase activities, reflecting an overall increase in energy reserve. Treatment with the calcium channel antagonists verapamil and nifedipine produced a significant increase in adenylyl cyclase activity and beta-adrenergic receptor density. Nifedipine treatment resulted in upregulation of both beta-adrenergic receptors and dihydropyridine receptors. This finding was associated with enhanced peak twitch force at all extracellular Ca2+ concentrations. We demonstrate for the first time that clinically used pharmacological agents (nifedipine and propranolol) result in alteration in two transmembrane signaling pathways, with associated alterations in physiological performance. Moreover, agents without cardioprotective effect in furazolidone-induced cardiomyopathy did not induce alterations in transmembrane signaling or energetics in normal hearts.

Myocardial Ca-sequestration failure and compensatory increase in Ca-ATPase with congestive cardiomyopathy: kinetic characterization by a homogenate microassay using real-time ratiometric indo-1 spectrofluorometry

A novel, simple, rapid and reproducible microassay is used for kinetic analysis of Ca-sequestration by homogenates of myocardium of turkeys with furazolidone-induced congestive cardiomyopathy. The assay monitors Ca in real-time using dual-emission ratiometric spectrofluorometry and the Ca-indicator dye indo-1. Using this assay and isolated SR studies we make several novel findings regarding the mechanism of SR failure in furazolidone cardiomyopathy. Qualitative differences in Ca-sequestration were not detected between groups. However, compared to controls the furazolidone treatment resulted in: 1) 50% depression in maximal activities (1.54 +/- 0.36 vs 0.73 +/- 0.12 microM/sec); 2) 2-fold increases in post-sequestration concentrations of ionized Ca (79 +/- 23 vs 141 +/- 13 nmol Ca/L homogenate); 3) 2-fold increases in Ca half-life (415 vs 790 msec); and 4) 25% increased passive Ca-binding capacity of homogenates. The Ca-ATPase specific activity of isolated sarcoplasmic reticulum was 60% increased in congestive cardiomyopathy (543 +/- 140 vs 873 +/- 108 nmol ATP hydrolyzed/min/mg membrane protein) although membrane yield was 20% decreased (0.79 +/- 0.09 vs 0.63 +/- 0.03 mg/g heart). The increased ATPase and decreased Ca-uptake activities in combination with the occurrence of 36% cardiac hypertrophy and 19% decreased body weights resulted in estimates of the relative energy cost to the animal for myocardial Ca transport being 5.5-fold increased with cardiomyopathy (20.5 vs 111 nmol ATP hydrolyzed per microM decrease of sarcoplasmic free Ca/kg body weight). These data indicate that congestive cardiomyopathy is associated with markedly increased permeability of sarcoplasmic reticulum to Ca and compensatorily increased Ca-ATPase activity. Accelerated energy consumption due to the increased energy cost of Ca transport and increased time of myocyte activation are predicted to predispose the myocardium to fatigue and irreversible failure.

Morphological changes associated with furazolidone-induced cardiomyopathy: Effects of digoxin and propranolol

The purpose of the present study was to examine light microscopic data qualitatively as well as quantitatively from an animal model of dilated cardiomyopathy in the turkey. A previous study reported the gross cardioprotective effect of propranolol and the lack of cardioprotection with digoxin in furazolidone-induced cardiomyopathy. It was therefore important to define whether the response of the myocardium to therapeutic interventions differed from the structural responses seen in their absence. In furazolidone-treated birds with resultant cardiac dilation there was myocyte hypertrophy, enlargement of nuclei and reorientation of subepicardial myocardial fibres. Similar changes were noted in birds receiving both furazolidone and digoxin. However, birds receiving propranolol, a non-selective beta-receptor antagonist, and furazolidone did not demonstrate a hypertrophic response or reorientation of fibres. These data indicate that propranolol maintained myocardial morphology and morphometry and thus prevented the structural sequelae of the disease, which is a better achievement of therapy than simple arrest of the progress. A role for intracellular calcium is implied. The cardioprotective effect seen with beta blockade suggests that membrane related events may lead to the contractile dysfunction that results in dilation and cardiac hypertrophy.

Myocardial biochemical changes in furazolidone-induced cardiomyopathy of turkeys

This study tested the hypothesis that membrane transport is the major biochemical system of the myocardium altered in furazolidone-induced cardiomyopathy (round heart disease), before the development of myocardial failure, and that metabolic enzymes and contractile proteins are less affected. Compared with controls, maximal percentage depression of activities of myocardium from furazolidone-treated birds were 40 for creatine kinase, 30 for glycolysis, 30 for glycogen, 20 for myofibrils, 20 for Krebs's cycle enzymes, 15 for fatty acid oxidation and 10 for total soluble protein. Sodium and potassium transport, antioxidant system activity, myosin, myosin isoenzyme patterns and amino acid aminotransferases were unaffected. In marked contrast, the calcium-transport ATPase activity of the sarcoplasmic reticulum had undergone a 60 per cent compensatory increase in activity. The pattern of biochemical changes observed is consistent with a role of ischaemia in the pathogenesis of round heart disease and indicates that calcium transport by the sarcoplasmic reticulum is the major biochemical system affected.

Influence of thiamin supplements on furazolidone-induced cardiomyopathy in turkey poults

Furazolidone (700 ppm) was fed to turkey poults from 2 to 5 weeks of age. The drug produced a cardiomyopathy and reduced the feed intake and growth of the birds. Thiamin was concurrently injected into the furazolidone-fed poults to determine whether the vitamin would prevent or reduce the severity of the cardiotoxic effect of the drug. Supplemental injections of thiamin had no significant effect on feed consumption or growth of the birds nor did they protect the heart against the cardiotoxicity. The conclusion is that furazolidone-induced cardiomyopathy is not caused by a thiamin deficiency.

Quantitative morphological alterations during the development of furazolidone-induced cardiomyopathy in turkeys

Cardiac structures were measured to determine the sequence of alterations during the development of spontaneous and furazolidone-induced cardiomyopathy in turkey poults 20 to 31 days after hatching. Samples of tissue from the free walls of the ventricles were removed and processed for electron microscopy. Volumetric density of cardiac structures was determined by the point-counting technique. The results indicate that the sequence of myocardial alterations is similar in both spontaneous and furazolidone-induced cardiomyopathy, but that these changes are more pronounced in the latter condition. Major changes include an early diminution in myofibrillar density, accompanied by a small reduction in mitochondrial density. This is followed by dilatation of the ventricular lumina and a decrease in the volume fraction of Z-bands. The increase in mass of the free walls of the ventricles in affected poults is due primarily to an increase in extracellular components and sarcoplasm devoid of organelles. The results suggest that the development of this myopathy is initiated by damage to the myofibrils of the cardiocytes.

Changes in myocardial ultrastructure during development of furazolidone-induced cardiomyopathy in turkeys

Ultrastructural alterations were studied in spontaneous round heart and furazolidone (FZ)-induced cardiomyopathy in turkey poults 20 to 31 days after hatching. The two most consistent morphological changes were abnormal accumulation of cytoplasmic glycogen and myofibrillar lysis. These alterations were more extensive in the drug-induced condition, which suggests that more myocytes are affected than in the spontaneous form of the disease. Data indicate that the myocyte contractile proteins sustain the major injury while other organelles appear to be involved only in an incidental manner. These findings corroborate previous suppositions which pointed toward a metabolic defect as the basic mechanism underlying the development of both spontaneous round heart and FZ-induced cardiomyopathy in turkey poults.

Protection of turkeys against furazolidone-induced cardiomyopathy

When fed furazolidone, 700 ppm, with their mash, most turkey poults develop dilated cardiomyopathy characterized by gross left ventricular dilatation with thinning of both the left ventricular free wall and ventricular septum. Birds fed propranolol, but not digoxin, did not develop this cardiomyopathy. It is not known what pharmacologic property of propranolol conferred protection or if mammals would receive similar protection.

Monoamine Oxidase Inhibition and Furazolidone-Induced Cardiomyopathy in Turkey Poults

Furazolidone (FZ), a potent inhibitor of monoamine oxidase (MAO), fed at concentrations of 700 ppm to turkey poults 2 through 5 weeks of age, produces cardiomyopathy and cardiomegaly. To determine the role of MAO inhibition in FZ-induced cardiomyopathy, the effects of FZ were compared with those of a chemically different MAO inhibitor, tranylcypromine (TCP), and a chemically similar non-MAO inhibitor, nitrofurazone (NFZ). At 14 days of age, poults were divided randomly into four groups. Control poults were fed a standard turkey starter ration, and poults in the experimental groups were fed the standard ration that included one of the following drugs at the given concentrations: 700 ppm FZ, 350 ppm NFZ, or 50 ppm TCP. Activity of MAO was assayed in liver and kidney homogenates from poults chosen at random at 14, 17, 21, 28, and 35 days of age in one trial and on heart homogenates at 14, 15, 17, 19, and 28 days of age in another trial. Development of cardiomyopathy was assessed using an electrocardiographic (ECG) technique and substantiated with necropsy examination. Inhibition of MAO was observed only in the heart in FZ-fed poults, only in the kidney in NFZ-fed poults, and in all three tissues studied in TCP-fed poults. Under the conditions of this study, an increase in the incidence of cardiomyopathy, based on ECG data and necropsy results, occurred only in the FZ-fed group. The inability of the potent MAO inhibitor, TCP, to produce cardiomyopathy in this study supports the conclusion that the primary mechanism of FZ-induced cardiomyopathy in turkey poults is probably not due to MAO inhibition.

Myocardial calcium levels in furazolidone-induced cardiomyopathy in turkey poults

1. Myocardial calcium levels were measured in control turkey poults and in poults with furazolidone (FZ)-induced cardiomyopathy. 2. FZ at a dose of 700 ppm added to the feed of poults from 2 to 5 weeks post-hatching produced the typical round-heart syndrome but had little effect on the calcium levels in the myocardium. 3. Data suggest that the mechanism of FZ cardiotoxicity is not associated with calcium overload.

Ultrastructural Changes of Sodium Chloride-Induced Cardiomyopathy in Turkey Poults

Round heart disease and ascites were induced in 8-day-old turkey poults by administering 0.75% sodium chloride in distilled water. Clinical signs included increased water consumption, loose feces, depression, and increased respiratory rate. Mortality was noticed after 3 days on the saline solution. Light microscopic and electron microscopic studies were made on the right and left free cardiac ventricular walls. Massive glycogen accumulations were demonstrated by periodic acid-Schiff staining and malt diastase digestion. Electron microscopic studies further showed the presence of extensive intrafibrillar glycogen, myofibrillar disarray, lysis of myofilaments, Z-band streaming, and disruption of intercalated discs. These changes are consistent with congestive heart failure and are similar to furazolidone-induced cardiomyopathy. A mechanism is suggested whereby sodium ions may cause hypertension and also act directly to influence the electrical and contractile activity of the heart muscle.

Role of taurine in furazolidone-induced cardiomyopathy

1. Taurine levels were determined in the heart, brain, and red and white muscle of turkey poults fed furazolidone (FZ) at a dose of 700 ppm from 2 to 5 weeks of age and/or 0.1 M taurine from 1 to 5 weeks of age. 2. In FZ-fed poults, taurine levels in the myocardium decreased significantly ( P < 0.001), but not in brain or muscle indicating a specific effect of FZ on the heart. 3. Exogenous administration of taurine significantly ( P < 0.001) increased taurine content of brain and muscle in all poults. 4. Myocardial taurine levels were significantly elevated ( P < 0.01) in poults fed a normal ration, but not in those fed FZ. 5. Although taurine treatment restored myocardial taurine to normal levels in the FZ-fed poults, the treatment did not decrease the incidence of FZ-induced cardioyopathy. 6. Since taurine is a membrane stabilizer and protects against calcium overload, our data suggests that sarcolemmal damage leading to tissue calcium overload is not the mechanism of the furazolidone-mediated syndrome.

Cardiac taurine levels and sarcolemmal calcium binding activity in furazolidone-induced cardiomyopathy

1. Cardiac taurine levels and sarcolemmal calcium binding activity were investigated in control turkey poults and in poults with furazolidone (FZ)-induced cardiomyopathy. 2. FZ at a dose of 700 ppm added to the feed of poults from 2 to 5 weeks post-hatching significantly reduced cardiac taurine levels and elevated the sarcolemmal calcium bindingactivity at high calcium levels. 3. The effect of FZ on cardiac taurine levels was reversed by removing the drug from the diet for a period of 2 weeks. 4. Data suggests that the cardiotoxicity of FZ is associated with severe damage to the sarcolemma.

A Morphometric Study of Myocardial Mitochondria and Myofibrils in Turkey Poults during Development of Furazolidone-Induced Cardiomyopathy

Furazolidone (FZ) at a dose of 700 ppm was fed to turkey poults 2-5 weeks posthatching. At 3, 4, and 5 weeks of age, six poults each were sacrificed from control and FZ-fed groups. Samples of tissue from the outer free walls of the ventricles and the interventricular septum were removed and processed for electron microscopy. Volumetric density of mitochondria and myofibrils was determined by the point-counting technique. Significant reductions (p less than or equal to .05) in the volumetric density of mitochondria were observed at 5 weeks of age in the ventricular free walls of round-heart poults, and at 4 and 5 weeks of age in the interventricular septum. Significant reductions (p less than or equal to .05) in the volumetric density of myofibrils were noted at 4 weeks of age in the left ventricular free wall of FZ-fed normal poults and in the interventricular septum of round-heart poults. At 5 weeks of age, a significant increase (p less than or equal to .05) in myofibrils was observed in the left ventricular free wall of FZ-fed normal poults. Data confirms that the volumetric densities of mitochondria and myofibrils are significantly altered during development of FZ-induced hypertrophy.

Distribution of Myocardial Glycogen in Turkey Poults During Development of Furazolidone-Induced Cardiomyopathy

Furazolidone (FZ) at 700 ppm was added to feed mixtures fed turkey poults two weeks posthatching to induce experimental cardiomyopathy. Poults in the control pen received the same ration but without FZ. From ECG data obtained at weekly intervals, poults were selected for sacrifice at 3, 4, and 5 weeks of age. Myocardial samples from the right and left ventricular free walls were analyzed for glycogen by biochemical assay technics. Levels of glycogen were significantly (P<.01) elevated in the FZ-treated poults at 3, 4, and 5 weeks of age. At all ages, the increase occurred in both the acid-soluble (TCA) and acid-insoluble (KOH) fractions with a proportionately greater increase in the TCA fraction. Results suggest that the effect of FZ on myocardial glycogen metabolism occurs very early and is more pronounced in the right ventricle than in the left ventricle even though both chambers may exhibit similar gross changes.

Furazolidone-Induced Cardiomyopathy: Biomedical Model for the Study of Cardiac Hypertrophy and Congestive Heart Failure

Furazolidone-Induced Cardiomyopathy: Biomedical Model for the Study of Cardiac Hypertrophy and Congestive Heart Failure

Characterization of glycogen in selected tissues of turkey poults with spontaneous round heart disease and furazolidone-induced cardiomyopathy

Furazolidone (FZ) at 700 ppm was added to feed mixtures fed turkey poults 2--5 weeks after hatching to induce acute experimental cardiomyopathy. Poults in the control pen received the same ration but without FZ. From EKG data obtained at weekly intervals, poults were selected for sacrifice at 5 and 10 weeks of age. Poults were sacrificed by cervical dislocation and appropriate samples of tissue from the left ventricle, liver, pectoralis and tibialis cranialis muscles were removed for glycogen assays. Character of glycogen, as determined by percent of branching and number of glucose units per segment, was not significantly altered in poults with spontaneous round heart disease or FZ-induced cardiomyopathy. This suggests that the glycogen accumulation noted in these conditions most closely resembles type II glycogenosis.

Blood Glucose and Tissue Glycogen Levels in Turkey Poults with Spontaneous Round Heart Disease and Furazolidone-Induced Cardiomyopathy

Furazolidone (FZ) at 700 ppm was added to feed mixtures fed turkey poults two weeks posthatching to induce acute experimental cardiomyopathy. Poults in the control pen received the same ration but without FZ. Four of the control poults developed spontaneous round heart disease. From EKG data and blood samples obtained at weekly intervals, poults were selected for sacrifice at 5 weeks of age. Tissue samples from the left myocardial wall, liver, and pectoralis major and tibialis anterior muscles were analyzed for glycogen by biochemical assay. Blood glucose was determined with the Technicon autoanalyzer. Deposition of glycogen increased significantly (p less than 0.05) in the myocardium of all affected poults and in the liver of all FZ-treated poults. Glycogen levels of the pectoralis major and tibialis anterior muscles were not affected by FZ, but a significant increase (p less than 0.05) was apparent in the pectoralis major muscle of spontaneous round heart poults. It was concluded that FZ influences glycogen metabolism, probably by enzyme inhibition, and that it tends to magnify effects seen in the spontaneous round heart syndrome. Glycogen infiltration of tissues such as the heart and white skeletal muscle suggests that the round heart syndrome may be a manifestation of the glycogen storage disease, idiopathic generalized glycogenosis. Lack of significant differences in the blood serum glucose levels of all poults indicates that these levels are not a reliable clinical parameter for monitoring development of the round heart syndrome.