TROPONIN T AND HISTOLOGICAL CHARACTERISTICS OF RAT MYOCARDIAL INFARCTION INDUCED BY ISOPROTERENOL

TIn our investigation,we used short-time model of myocardial infarction of rats induced by high dose of isoproterenol (ISP). We investigated cardiac troponin T blood level (cTnT) and histological characteristics of rat myocardium. ISP, single, intraperitoneal dose  mg/kg was given to male, adult, Wistar rats (n=). Rats were distributed depending on their body weight in subgroups: ISP I (BW -g) and ISP II (BW -g).Control group (n=) was treated with intraperitoneal dose of , NaCl. Cardiac TnT was measured by electrochemiluminiscence (ECLA) sandwich immunoassay in rat serum  hours after ISP application. Rats’ hearts were dissected and examined by qualitative histological method (HE). Statistical signifi cance was set at ,. Th ere was signifi cant diff erence in cTnT of ISP II (p=,) vs. control and ISP I (p<,) vs. control. Signifi cant diff erence was beetween ISP I and ISP II subgroups (p<.).Th e accent of histological changes of myocardium was on nuclei of cell. Cells showed acydophilic changes and nuclei disappearance as signs of coagulative necrosis development. Extensivity of histological changes were diff erent beetween ISP I and ISP II subgroup. Used dose of ISP induced development of myocardial necrosis in rats. Subendocardial portion of myocardium was more vulnerability than subepicardial portion. Rats of ISP II had more extensive histological changes than these in ISP I. Administered doses of ISP enabled cTnT utilization as a marker of myocardial necrosis.


Introduction
We're witnesses of permanent changes in diagnosing and therapy of acute myocardial infarction patients.Permanent changes are result of experimental investigations and better understanding of molecular mechanisms occurred during myocardial necrosis development.Myocardial infarction can be induced chemically and non-invasivelly in small laboratory animals like rats.Commonly used non-invasive techniques for induction of rat myocardial necrosis are those with use of catecholamines.For this purpose, isoproterenol-synthetic catecholamine is the most often used.It's β-adrenergic receptors agonist and causes severe stress in myocardium resulting in infarct-like lesion.Rona and coworkers published the fi rst results about ISP cardiotoxic eff ects ().Rat myocardial changes induced by ISP are a similar to human myocardium changes during myocardial infarction ().ISP produces a relative ischemia or hypoxia because of myocardial hyperactivity, coronary hypotension and cytosolic Ca + overload.Th ere is evidence that ISP cardiotoxicity is result of catecholamine oxidation into aminochromes ().Degree of pathomorfological changes depends on used ISP dose ().Changes are present in subendocardial portion of myocardium, apex, left ventricle, papilar muscle and close to coronary artery.Troponins are highly sensitive and specifi c markers of cardiac cell damage ().Th ese contractile proteins are released from myocardium in proportion to the degree of tissue injury.Cardiac troponin T (cTnT) belongs to the proteins of contractile apparatus that are unique for cardiac muscle.Except in myocardial infarction diagnosing, troponins should be included in the evaluation of cardiotoxicity and cardioprotective properties of new drugs ().Th ere is so little information about cTnT blood level and histological fi nding of myocardium in ISP induced rat model of myocardial infarction.There isn't wellstandardized short time animal model of rat myocardial infarction induced by ISP for studying of: a) histological and cardiac marker changes during myocardial infarction, b) cardioprotective and c) cardiotoxic drugs investigation.In this short time rat model, we examine histological characteristics of cardiac muscle damage and TnT blood level in rats  hours after ISP administration.

Material and Methods
We used twelve, adult, male, Wistar rats as experimental group.Th ey were raised and housed in air-conditioned, humidity-controlled cages.Rats had free access to water and commercial food during experimental period.Ethi-cal Committee of our Institution approved the experiment.Rats were distributed depending on their body weight in subgroups: ISP I (BW -g) and ISP II (BW -g).Rats of both subgroups were treated with single intraperitoneal (i.p.) dose of ISP ( mg/ kg BW).Control group (n=) was treated with intraperitoneal dose of ,  NaCl.Ketamin anesthesia (, ml/g BW) was performed in rats before of ISP administration.Isoproterenol hydrochloride was manufactured by Sigma Chemical Company, USA.Rats of ISP II died in diff erent intervals from ISP application within  hours.Blood of these animals was taken by cardiac punction.Rats of ISP I survived  hours of experimental period and blood samples were drawn from tail wein.Th e blood was centrifuged for  minutes at  r.p.m.Th e sera were frozen and stored at -o until determination.Th e determination of cTnT was performed with Elecsys, electrochemiluminiscence (ECLIA) sandwich immunoassay, manufactured by Roche Diagnostics.We used analyzer Elecsys , Roche.Values of cTnT are given in ng/ml.Th e chest cavities of the rats were opened to remove the heart shortly after blood samples were taken.Rats' hearts were dissected for histological examination.Left ventricular tissue was placed in  buff ered formalin solution, embedded in paraffi n, sectioned at  μm intervals and stained with hematoxylin-eosin (HE).Histological analysis was obtained by using microscope Nikon type E with installated digital camera.Results of histological analysis are presented by using qualitative histological analysis.Cardiac TnT blood level data were analyzed by two-tail, unpaired Students't-test.Significance was set at , .Results are reported as mean ± SD.

Results
Control rats were treated with saline and  hours after that blood values of circulated cTnT were minimal.
Mean value was ,  ng/ml.Four hours of experimental period after ISP aplication, rats of ISP I subgroup were survived and ISP II died in diff erent intervals.Compared to control rats, cTnT blood level of ISP treated rats was signifi cantly higher (p<,).Mean value was , ng/ml (Figure ).Signifi cance of diff erence of cTnT level in ISP I vs control was p<,.ISP II had diff erence at higher level of signifi cans p=,.Mean values of cTnT in ISP I were , ± S.D.,  ng/ml and for ISP II subgroup TnT was , ± , ng/ml.There was significant difference beetween ISP I and ISP II subgroups ( p<,) (  Rats of ISP II died in diff erent intervals from ISP application within  hours.Large blood vessels involved in subendocardial tissue are strong dilated (Figure ).Histological changes were more extensive compared to ISP I. Subendocardial portion is presented in form areas of massive necrosis, cellular arrangement disappeared, nuclei disappeared or in form of massive vacuola.In contrast to ISP I, rats of ISP II showes changes in subepicardial myocardium.Variable degree of nuclei cariolysis in subepicardial myocites is presented.

Discussion
In this study we used rat model of acute myocardial infarction induced by ISP.Based on Rona and coworkers experiences, it's known that ISP has harmful infl uence on heart.O'Brien and associates () have shown that troponins are a powerful biomarker in laboratory animals for sensitive and specifi c detection of cardiac injury arising from various causes.From earlier ISP stud-ies in rats, it is known that high doses of ISP (- mg/ kg) induce acute myocardial damage that has consequence in increasing of blood cTnT ().We knew that cTnT determination with the cTnT assay, which was developed originally for human sera, was possible in the rat with the monoclonal antibodies used, because of cross-reaction between humans and rats cTnT ().Very small amount of cTnT circulates as result of natural protein turnover.Minimal circulating cTnT values were obtained in our control rats.Detection of circu-lating cTnT depends on assay sensitivity.Control rats had TnT mean value ,  ng/ml.Intraperitoneal, single ISP dose, administered to rats caused expectant, significant increasing cTnT.From damaged myocites, fi rst is released free citosolic pool of cTnT.After that portion, cTnT bounded in contractile apparatus is released by proteolytic degradation.We get signifi cant diff erence in cTnT blood levels between ISP II and I subgroups (p<,) as consequence of diff errence in extensivity of histological changes.Th ese results are in accordance to obtained results of qualitative histological analysis.Degree of myocardium pathomorfological changes depends on used ISP dose ().Subcutaneous injection of ISP  mg/kg causes rat myocardial necrosis due to prolonged tachycardia.Th e proposed mechanism for ISP myocardial necrosis are myocardial hypoperfusion, glycogen depletion, electrolyte imbalance, lipid accumulation and free radical damage (,).According to Preus and coworkers,  hours after ISP application, cells showed acydophilic changes and nuclei disappearance as signs of coagulative necrosis development ().Th ere aren't published data about using higher doses of ISP and expressed myocardial changes in fi rst hours after application.By using ISP  mg/kg dose, we noted extensive myocardial lesions  hours from drug application.Results of histological analysis gave us clear evidence of necrogenic eff ect of ISP on heart.Coagulative necrosis induced by ISP, which we found in our study, is the same in case of heart damage caused by ischaemia and presented in human myocardial infarction.Massive myocardial necrosis characterizes human myocardial infarction while catecholamine model of myocardial necrosis is presented in form of focal coagulative necrosis.Joseph and Balasz described focal and multiple areas of myofi brillar necrosis after ISP application to rats ().Our results are in concordance with their fi ndings.Histological examination of the myocardial tissue in all ISP treated rats showed necrotic areas in the subendocardial layer of the left ventricle.Poorer vascularisation and oxygen supply of subendocardial portion is possible cause of more vulnerability.By qualitative histogical analysis, we noted the diff erence in extensivity of myocardial changes between ISP I and ISP II rats.Rats of ISP II had higher body weight (mean BW  g) than ISP I rats (mean BW g).ISP I subgroup have had changed subendocardial portion of myocardium, thus subepicardial portion didn't show any changes.Lesions were presented in form of widespread focal necrotic areas.Cardiomyocites were most frequently without nuclei, lost of the normal myofi brilar striation pattern.Th ere were variable degrees of damage.Rats of ISP II had expressed myocites changes in subepicardial and subendocardial portion of myocardium.Subendocardial alterations were more extensive than in ISP I subgroup.Th ere were signs of massive necrosis with disappeared cellular arrangement and lost edematous and fragmented myocites.Large blood vessel in myocardium of ISP II group were dilated greatly and injected which present the sign of shock development and myocardial hypoperfusion.Cells' nuclei have disappeared or presented in form of massive vacuola.Sarcoplasma of cells were fragmented.ISP II rats have had altered subepicardial portion of myocardial tissue in comparing with ISP I. Subepicardial myocites had variable degree of kariolysis.Sarcoplasma of isolated myocites showed separation and vacuolization of myofi brils.Lipolysis due to the adrenergic action of ISP is a potential factor in ISP myocardial necrosis development.Th e heart suplies a signifi cant portion of its fatty acid substrates as free fatty acids derived by lipolysis from adipose tissue.Although lipid availability is important for the heart, excess level of fatty acids in cardiomyocites can be deleterious.According to Mohan, lipids mobilized from the adipose depot reach their highest level in blood one hour after ISP administration and are cleared by  hours ().Lipolysis as leading critical biochemical event in induced ISP myocardial necrosis does not occur until  hours after ISP injection, and this time frame sugests a critical role for lipolysis.Possible mechanism of diff erence in extent of histological changes between ISP II and ISP I subgroup is lipolysis.

Conclusion
Dose of ISP ( mg/kg) induced myocardial necrosis development in rats.Subendocardial myocardium showed more vulnerability compared to subepicardial portion.Rats of ISP II had more extensive myocardial changes than these in ISPI.Obtained results pointed at possible additive influence of lipolysis on cardiotoxic effects of ISP.Administered doses of ISP enabled cTnT utilization as a marker of myocardial necrosis.This animal model of myocardial infarction is suitable for cardiotoxic and possible cardioprotective substances investigation.
Figure .(A and B)  presents normal myocardial tissue of control rats.Th e myocardium is composed of muscle fi bers.Each fi ber is enclosed by a sarcolemma.Th e nuclei are generally located in the central portion of the fi ber.A net of reticular fi bers and fi ne collagenous fi bers surround each muscle fi ber.Th e myocardium is richly supplied with small vascular channels forming an intramural circulation.Subendocardial myocardium of ISP I rats is presented with areas of necrotic changed myocytes.Myocites show variable degree of damage and accent of pathological changes are on nuclei.Cells are edematous, lost of the normal striation pattern and posses nuclei with inhomogeneous content(Figure .).Deposited material of hyperacidophilic features are presented close to sarcolemma (.B).In necrotic myocites myofi brillar lysis is complete.Deep subendocardial portion showes diff use arranged focal necrosis of myocites.Nuclei of these areas are in phases of picnosis or lysis or completely disappearance.Blood vessels near necrotic area contain eosinophilic amorfi c material.Subepicardial myocardium of ISP I not deviate from normal myocardial tissue of control rats.