Cardiac Troponin I and T Alterations in Dog Hearts With Myocardial Infarction Correlation With Infarct Size

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1 Cardiac Troponin I and T Alterations in Dog Hearts With Myocardial Infarction Correlation With Infarct Size Vincent Ricchiuti, and FredS. Apple, PhD, Scott W. Sharkey, MD, Mary Ann M. Murakami, Ellen M. Voss, MS, PhD Key Words: Cardiac troponin I; Cardiac troponin T; Acute myocardial infarction; Ventricles; Infarct size Abstract We studied the distribution of cardiac troponins I (ctnl) and T (ctnt) in ischemic left ventricular (LV) tissue in 7 infarct zones, 7 remote nonischemic LV areas, and 7 nonischemic areas each from the right ventricle and circumflex in an acute coronary artery occlusion dog model to correlate myocardial loss of troponins with infarct size 3 weeks after the infarction and to determine whether the decrease of troponins in ischemic myocardium can be used to assess the infarct size in dogs after coronary occlusion. The serum profiles for time vs mean ctnl and ctnt concentrations in 6 dogs after occlusion showed peak concentrations at day and 5 days, respectively. The concentrations of troponins were similar in all nonischemic zones. However, ctnl and ctnt decreased significantly in the LV ischemic tissues. Loss ofctnt, but not ctnl, in ischemic LV tissues correlated significantly with infarct size 3 weeks after the infarction. Biochemical alterations suggest that the increases in serum troponins after the infarction parallel the decreases in tissue concentrations of troponins. The extent of myocardial necrosis after acute myocardial infarction (AMI) is an important determinant of the risk of death.2 Identification of a tissue marker of which release into the circulation bears a close relation to the degree of myocardial damage would be desirable. Such a marker should be more sensitive and specific than current markers, should provide prognostic information, and should be measurable in a time frame that permits treatment to minimize further necrosis.34 Troponins I and T (Tnl and TnT) are 2 proteins of the thin filament-associated regulatory system of the contractile complex of skeletal and heart muscle.5 Troponins I and T are expressed in 3 different isoforms, namely, slow- and fast-twitch skeletal muscle Tnl and TnT and cardiac troponin I (ctnl) and T (ctnt).6"9 The cardiac subunits of Tnl and TnT are encoded by a separate gene from skeletal isoforms giving them a unique amino acid sequence, allowing for monoclonal antibody development and commercial production of immunoassays for use in clinical laboratories.0-3 Published studies have demonstrated the improved usefulness of ctnl and ctnt for the diagnosis of AMI4-8 compared with creatine kinase MB, the most frequently used marker of AMI.9-22 Increased serum concentrations of ctnl and ctnt have been shown to be more tissue specific for myocardial damage than other cardiac markers Furthermore, since the majority of cardiac troponins are myofibril bound, their release in serum may correlate with the extent of cardiac necrosis after myocardial infarction, providing a noninvasive method for assessing infarct size. The similarity of the canine heart to the human heart in size and physiology has resulted in the use of dogs as an experimental model for cardiac disease and injury. It is important to understand the distribution of ctnl and ctnt in the normal dog heart and the effects of disease on changes Am J Clin Pathol 998;0:

2 Ricchiuti et al / DISTRIBUTION OF CARDIAC TROPONIN I AND T IN DOG HEARTS in their distribution in ischemic myocardium. Differences in distribution within different areas of the heart could have an impact on the ability of the troponin markers to accurately indicate the presence of injury and the extent of damage to myocardial cells. The purposes of this study were to () examine serial ctnl and ctnt release into blood during a 3week period after acute coronary artery occlusion; (2) determine the distribution of ctnl and ctnt in ischemic and nonischemic myocardium associated with AMI; and (3) determine whether the decrease of ctnl and ctnt in ischemic myocardium can be used to assess the infarct size in dogs after coronary artery occlusion. Materials and Methods samples were collected, no US Food and Drug Administration-approved ctnl immunoassays were available. In this study, immunoassays of ctnl mass in the described serum was performed with an analyzer (Stratus II, Dade, Miami, Fla) that uses 2 ctnl-specific monoclonal antibodies as capture and labeled antibody.9-30 Less than 0% variability between fresh samples and samples kept frozen at -40 C for 8 months was demonstrated (data not shown). The reference serum levels for canine ctnt and ctnl were established from serum samples of 0 control dogs. The reference ranges of the serum concentrations obtained for ctnt and ctnl were similar to human reference ranges. Consequently, the upper reference limits for ctnt and ctnl in dogs were similar to those recommended by the manufacturers for human assays. The upper reference limit for ctnt in dogs was established at 0. u.g/l. The upper reference limit for ctnl in dogs was established at 0.6 J.g/L. Study Design The acute myocardial infarction model used in this study has been described previously.26~29 Briefly, mongrel dogs of either sex weighing 20 to 30 kg were anesthetized with intravenous sodium pentobarbital, 30 mg/kg, intubated, and ventilated with a volume respirator. A polyvinyl catheter was inserted via a right femoral artery cutdown for the purpose of monitoring blood gases and left ventricular (LV) pressures. A midline sternotomy was performed, and the heart was suspended in a pericardial cradle. Lidocaine was administered as prophylaxis against ventricular arrhythmias. A short segment of the mid-left anterior descending coronary artery was dissected free of the surrounding epicardium and coronary vein. Seven dogs underwent surgical coronary artery occlusion resulting in LV infarction, and 3 had no coronary artery occlusion (controls). After chest closure, the animals were placed into observation during a 3-week period. Animals were killed by injection of 400 meq of potassium chloride solution. The heart was immediately removed and the atria excised. The ventricles were cut into 4 cross-sections and placed into % triphenyltetrazolium chloride (TTC) for 20 minutes. The TTC-negative tissues were measured by planimetry and expressed as a percentage of LV weight and area as an index of infarct size. The sections were then preserved in 30% formalin solution. Quantification of ctnl This study was designed to collect myocardial tissue biopsy specimens and not blood samples. However, in a previous similar study,26 serum samples were collected from 6 dogs at the time of occlusion (baseline), daily for 4 days after occlusion, and at the time of sacrifice (3 weeks), and ctnt levels were measured. These experimental samples, since frozen at -40 C, were used for measurement of ctnl for comparison with ctnt findings. 26 At the time the 242 Am J Clin Pathol 998;0: Tissue Biopsy Specimens In this study, tissue from a new group (different dogs than in the previous study26) of dogs with occluded vessels (n = 7) was obtained from ischemic LV tissue in the infarct zones, remote nonischemic LV areas, and nonischemic tissue from the right ventricle (RV) and circumflex (Cx). For all the control dogs that had undergone sham operations (n = 3), tissue was obtained from nonischemic areas (LV, RV, and Cx). All biopsy specimens were immediately frozen at -40 C. The locations of biopsy specimens in relation to the area of necrosis were noted at the time of TTC staining.26 Biopsy sites were marked with colored pin heads, and the LV cross sections were photographed after TTC staining. This allowed correlation of TTC staining with ctnl and ctnt data. Protein Extraction Frozen heart samples (~ 50 mg) were coarsely ground in a liquid nitrogen-cooled mortar and added to ml of ice cold buffer (a 200 mmol/l concentration of potassium phosphate, ph 7.4, a 5.0 mmol/l concentration of ethyleneglycoltetraacetic acid, a 5.0 concentration of mmol/l 3-mercaptoethanol, and 0% [vol/vol] glycerol) to release mitochondrial and cytoplasmic enzymes.29 The samples were homogenized at 4 C for 20 seconds at high speed with a tissue homogenizer (Polytron, Brinkman Instruments, Westbury, NY). This was followed by a -hour incubation at room temperature with gentle agitation and subsequent centrifugation at 40,000g for 30 minutes at 4 C. The procedures resulted in more than 98% recovery of both cytosolic and myofibril proteins.26 The supernatants were used immediately in biochemical analysis and Western blot analysis. Western blot analysis of the samples during the 2-month experimental period did not show any degradation of the ctnt or ctnl proteins. The protein concentration was determined by using

3 a modified Lowry method3 (Sigma Diagnostics, St Louis, Mo) with bovine serum albumin as a standard. Antibodies Two different primary antibodies were selected for use in the Western blot analysis based on preliminary tests that characterized antibody specificity using purified human ctnl and ctnt proteins (gift from Spectral Diagnostics Inc, Toronto, Ontario). A mouse monoclonal antibody specific for ctnl (E2)32 was a gift from Sanofi Diagnostics Pasteur-ERIA, Lyon, France, and was used at 2 ug/ml. The mouse monoclonal antibody specific for ctnt (designated as JS-2),33 was a gift from Lakeland Biomedical, Minneapolis, Minn, and also was used at 2 (ig/ml. Western Blot Analysis Protein extracts (5 Lig) were size-fractionated on 2% sodium dodecyl sulfate-polyacrylamide gels34 and subsequently transferred to nitrocellulose membranes (Hybond, Amersham, Arlington Heights, ).35 Nonspecific binding sites were blocked by incubating the membranes in a blocking buffer (5.0% nonfat dry milk and TTBS [a 20 mmol/l TrisHC, ph 7.6, a 37 mmol/l NaCl, and 0.% Tween 20]) for hour. The primary antibody was diluted in antibody buffer (.0% nonfat dry milk, TTBS) and incubated with the membranes for 2 hours on a rotating cylinder. The membranes were washed three times in changes of TTBS buffer for 30 minutes. Appropriate horseradish peroxidase-labeled secondary antibodies (sheep antimouse) were then incubated with the membranes for hour. The membranes were again washed 3 times in TTBS buffer before a -minute incubation with a chemiluminescent substrate (ECL, Amersham). Light emission was detected by exposure to Fuji RX autoradiography film Fujifilm, Tokyo, Japan) in the presence of Cronex (Fisher Scientific, Chicago, ) intensifying screens. Signal intensities within the linear range were measured by using laser densitometry (Molecular Dynamics Inc, Sunnyvale, Calif). Linearity was established by analysis of a standard curve generated with known amounts of total protein by Western blot analysis (data not shown). One control dog sample was transferred onto all the membranes, allowing for comparison of sample intensities and the normalization of all values between different membranes. Statistical Interpretation of Data Percentages of change were used to compare changes in tissue concentrations between ischemic and nonischemic tissues. Unpaired t tests and -way and 2-way analyses of variance were used to determine the significance of the changes; P<.05 was considered significant. Linear regression analysis was performed to compare infarct size with percentage of change of proteins in myocardial tissue. Results were expressed as mean (SD, SE, or 95% confidence intervals). Statistical analysis was performed on a Power Computing computer (Round Rock, Tex) using appropriate statistical software. Results Figure II shows the serum time vs mean ctnl (Figure, A) and ctnt (Figure, B) concentrations plotted as an increase above their respective reference limits in 6 dogs after coronary artery occlusion. For each protein, the profiles were similar for all 6 dogs. The serum profile of ctnl showed a monophasic peak on the first day, remained elevated for 7 days, and returned to baseline by 3 weeks after occlusion (Figure, A). In comparison, the serum profile of ctnt also showed a monophasic distribution, peaking at day 5 (Figure, B), remaining elevated for 7 days, and returned to baseline by 3 weeks after occlusion. In the present study involving a new group of dogs (n = 7, different animals than those used for the serum analysis shown in Figure ), Western blot analysis of cardiac troponin I and T levels from ischemic and nonischemic myocardium demonstrated migration of isoform to positions corresponding to approximately 29 and 39 kd, respectively, on 2% sodium dodecyl sulfate-polyacrylamide gels ( Image II). No cross-reactivity with troponin I or T skeletal forms or creatine kinase subunits was observed with mouse monoclonal antibody specific for ctnl or ctnt used in the Western blot studies. Linearity was verified using laser densitometry analysis of the standard curves generated with known amounts of antibodies (data not shown). IFigure 2 compares the cardiac distribution of ctnl and ctnt in ischemic and nonischemic tissues 3 weeks after coronary artery occlusion. Our results showed no differences in nonischemic LV, RV, and Cx tissues. Also no significant differences were observed between control heart tissues and the nonischemic tissues from the coronary arteries of dogs with occlusion (data not shown). Within the same hearts (n = 7), densitometry of Western blot autoradiograms showed ctnl and ctnt in ischemic LV tissue (n = 7) decreased 42% to 92% (P<.0) and 52% to 62% (P<.02), respectively, compared with nonischemic zones (n = 7). Infarct size, expressed as the percentage of the volume of infarct tissue, ranged from 5.% to 5.2% (mean [SD] 3.9% ± 2.3%). IFigure 3 shows the linear regression analyses for measurements of loss of ctnt and ctnl in infarcted tissue compared with infarct size. Linear regression analysis for loss of ctnt yielded the following equation: v = x, where y is the loss of ctnt in infarcted tissue (%) and x is the infarct size in the same tissue measured by TTC staining. The correlation coefficient Am J Clin Pathol 998; 0:

4 Ricchiuti et al / DISTRIBUTION OF CARDIAC TROPONIN I AND T IN DOG HEARTS d a d 200 I I H C Io I! i Days After Infarct Days After Infarct Figure I I A, Profile of mean (SD bars) concentrations of serum cardiac troponin I (ctnl) plotted as a function of increase above their respective upper reference limit (0.6 ug/l) vstime (days) from 6 dogs that underwent permanent surgical coronary artery occlusion. B, Profile of mean (SD bars) concentrations of serum cardiac troponin T (ctnt) plotted as a function of increase above the respective upper reference limit (0. iig/l) vs time (days) from 6 dogs that underwent permanent surgical coronary artery occlusion (adapted from Voss et al26). (Pearson r) was r = 0.90 (P<.05). For measurements of the loss of ctnl in infarcted tissue (%), the equation was y = x, with r = 0.36 (P<.54). These data indicate that the percentage of change of ctnt concentration in ischemic LV tissue was significantly correlated with infarct size, but the percentage of change of ctnl concentration was not. Discussion This study is the first to report myocardial distribution of ctnt and ctnl in ischemic LV tissue from a chronic AMI dog model. The main finding of this study demonstrates that after 3 weeks after myocardial infarction, there are significant decreases in the concentrations of ctnt and ctnl in ischemic LV tissue compared with nonischemic LV, RV, and Cx tissues (Image and Figure 2). Furthermore, loss of ctnt from ischemic LV tissue was significantly correlated with infarct size. Cardiac troponins I and T have attracted increasing interest in recent years as markers for myocardial infarction and damage because of their cardiac specificity.7 In a recent study involving a chronic left ventricle remodeling pig model,36 at 2 months after the infarction, Western blot analysis and biochemical assay showed that ctnl and ctnt in nonischemic remodeled LV tissue decreased 40% to 80% compared with normal tissue. The distribution of ctnt in normal and diseased human and 244 Am J Clin Pathol 998; 0: canine myocardium has been described previously.26 Release of ctnt and creatine kinase MB into serum after canine coronary artery occlusion was similar to that in humans. Parallel to the serum increases of ctnt, cytosolic and myofibril ctnt levels decreased in heart tissue after coronary artery occlusion in dogs and after AMI in humans. The clinical assumption is that the amount of these proteins appearing in the blood is proportional to the size of myocardial cell injury and necrosis. The results of the present study, based on direct quantification of ctnl and ctnt in an LV AMI dog model, are consistent with the previous reported alterations in ctnl and ctnt in an LV remodeling pig model.36 The consistency of the results in both models was interesting because the studies differed in their designs. The current study examined infarcted myocardium (dog model), while the previous study examined remodeled myocardium (pig model). Furthermore, in the pig model, loss of protein was from noninfarcted LV remodeled tissue, whereas the dogs underwent surgical occlusion of the left anterior descending coronary artery resulting in LV infarction, from which protein loss occurred. A recent study attempted to compare ctnl release with the results of other independent methods for quantifying infarct size in living patients. The authors demonstrated that ctnl release in patients with first-time myocardial infarction was significantly correlated with scintigraphic estimates of myocardial scarring.37 Cardiac troponin levels are known to increase in the blood after infarction; release and loss of

5 kd 6,000 ctnt ctnl 50.9 ctnt > mm mmm * * " Image I I Representative Western blots of dog heart protein extracts probed for cardiac troponin T (ctnt; top) and cardiac troponin I (ctnl; bottom) showing a decrease in the ischemic left ventricle (LV) (lane 2) compared with nonischemic LV (lane 3), nonischemic circumflex (lane 4), or nonischemic right ventricular (lane 5) dog heart tissue. Lane shows purified ctnt (top) and ctnl (bottom) proteins. Migration of molecular weight standards for each cardiac troponin is shown to the left. e D 2r 3,000 < 2,000,000 I LV Nl LV Nl Cx Nl RV Figure 2 Tissue distribution of cardiac troponin I (ctnl) and T (ctnt) in ischemic left ventricle (I LV) tissue, nonischemic left ventricle (Nl LV) tissue, nonischemic circumflex (Nl Cx) tissue, and nonischemic right ventricular (Nl RV) tissue. Arbitrary units represent relative intensity. Data are given as mean ± SD. Asterisk indicates P<.05 for ischemic LV compared with nonischemic LV dog heart tissue. troponins in chronic diseased hearts supports serum evidence of increased levels of ctnl and ctnt in patients with acute myocardial ischemia, unstable angina, or non-q-wave myocardial infarction and indicates a poor prognosis.3839 In contrast to these studies that provided indirect evidence, our study provides direct evidence for loss of ctnt from injured myocardium that relates to the size of myocardial damage. An AMI leads to chronic cardiac structural changes. These structural changes include hypertrophy of viable myocytes and chamber dilation.404 The degree of structural change is directly related to myocardial infarct size and is associated with clinical congestive heart failure.42 The myocardial biochemical alterations that accompany the structural changes remain unknown. An understanding of the myocardial molecular changes that occur after infarction and ischemia may lead to interventions designed to prevent the postinfarction remodeling process and subsequent clinical appearance of congestive heart failure.443 The implications are that appearance of ctnl and ctnt in the blood is due to ongoing release from diseased myocardium after coronary occlusion. Our observed biochemical alterations show that parallel to the serum increases of ctnl (and ctnt, Figure l26), ctnl and ctnt levels decreased in ischemic LV heart tissue after coronary artery occlusion in dogs compared with nonischemic LV, nonischemic RV, and nonischemic Cx heart tissue. the first day, an elevation for 7 days, and a return to baseline by 3 weeks after the occlusion (Figure A). In comparison, the serum profile of ctnt also showed a monophasic distribution with a peak on day 5 (Figure, B),26 an elevation for 7 days after the occlusion, and a return to baseline by 3 weeks after the occlusion (Figure ). While ctnl and ctnt are predominantly myofibril-bound24-26 with a minor cytosolic component, the explanation for the difference in serum time vs concentration profiles after AMI is not apparent. However the smaller size of ctnl (22,000 d) vs ctnt (40,000 d) could lead ctnl to be more easily lost from tissue after ischemia and necrosis during earlier phases of AMI after dissociation from the troponin I/C/T complex, giving rise to the earlier massive (or major) peak. Our results for the measurement by Western blot analysis of the loss of ctnl and ctnt from injured myocardium in dogs are consistent with the release of ctnl and ctnt in the blood measured by immunoassay as described. Indeed, we showed that ctnl and ctnt in ischemic LV hearts decreased 67% (P<.0) and 52% (P<.02), respectively, compared with nonischemic LV zones. Overall, these findings show that myocardial infarction is responsible for protein loss from ischemic myocardium, resulting in a nonuniform distribution of cardiac troponin subunits in the heart. Furthermore, measurement of the release of cardiac troponins in the circulation serves as a sensitive and specific indicator of myocardial injury. The measurement by immunoassay of serum ctnl concentrations showed a monophasic response with a peak on Certain limitations of the present study must also be addressed. First, different animals were used for blood and Am J Clin Pathol 998;0:

6 Ricchiuti et al / DISTRIBUTION OF CARDIAC TROPONIN I AND T IN DOG HEARTS 0 j Infarct Size (%) IFigure 3 Linear regression between the myocardial infarct size (reported as percentage of volume infarct of tissue, in abscissa) and the loss of cardiac troponins in infarcted tissue (expressed in percentages in ordinate). Two regression lines are drawn; they correspond with regression of myocardial infarction size with the loss in infarcted tissue of cardiac troponins T (black circle) and I (open circle). tissue measurements. Thus, measurements were not performed to quantitate ctnt and ctnl using blood from the experimental dogs (with coronary artery occlusion) to have a direct correlation with loss of proteins observed in the diseased myocardial tissue. However, the estimation of infarct size from analyses of serial serum cardiac troponin changes is based on the concept that myocardial troponin depletion is related quantitatively to the extent of myocardial necrosis. Cardiac troponin release from myocardium into serum has been observed to be associated with irreversible injury.26,3839 However, the measurement of infarct size at 3 weeks may introduce problems, since, by that time, the infarct may have been partially replaced by scar tissue occupying less volume than the original amount. It is plausible that the total myocardial loss of ctnl and ctnt could correlate with the total amount of ctnl or ctnt estimated to enter the bloodstream. In that case, the total ctnl or ctnt loss from the tissue requires measurement of how much ctnl or ctnt was lost per gram of infarct tissue multiplied by the amount of total estimated infarct tissue. Second, no Northern blot analysis was performed to measure the messenger RNA of ctnt and ctnl to correlate with the Western blot analysis protein profile. Finally, the study of ctnl and ctnt release from an infarct zone caused by permanent occlusion would be clinically relevant only for the subset of patients who do not receive thrombolytic therapy. The clinical implications of our findings suggest a chronic loss of cardiac troponin protein in remodeled myocardium, 246 Am J Clin Pathol 998; 0: potentially allowing for chronically increased serum ctnl and ctnt concentrations. While these findings might be misinterpreted as AMI,44 they would imply long-term release from injured myocardium. Studies have shown that patients with elevated serum cardiac troponin levels have more complicated cardiac courses associated with unstable coronary plaques.45,46 Blood studies involving patients with heart failure without AMI are necessary to assist the clinician in interpretation of serum cardiac troponin concentrations and patient treatment decisions. Our current efforts are designed to develop physiologically based models, to assess them experimentally, and to apply them to estimation and prediction of infarct size based on analysis of serial changes in ctnt and ctnl. Such models should be useful not only in broadening the applicability of ctnt and ctnl prediction of infarct size, but also in highlighting fruitful areas of investigation concerned with identifying processes governing the release of cardiac troponin protein and other biochemical markers from the ischemic heart and their behavior in the circulation. From the 'Departments of Laboratory Medicine and Pathology, Hennepin County Medical Center, and 2University of Minnesota School of Medicine, Minneapolis, Minnesota. Partial financial support of the research was received from Elf Aquitaine, Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France, and Upjohn, Kalamazoo, Mich. Address reprint requests to Dr Apple: Hennepin County Medical Center, Clinical Labs MC 82, 70 Park Ave, Minneapolis, MN References. Geltman EM, Ehsani AA, Campbell MK, et al. The influence of location and extent of myocardial infarction on long'tenn ventricular dysrhythmia and mortality. Circulation. 979;60: Rogers W], MacDaniel HG, Smith LR, et al. Correlation of angiographic estimates of myocardial infarct size and accumulated release of creatine kinase MB isoenzyme in man. Circulation. 977;56: Adams J III, Abendscheim DR, Jaffe AS. Biochemical markers of myocardial injury: is MB creatine kinase the choice for the 990s? Circulation. 993;88: Mair J, Morandell D, Genser N, et al. Equivalent early sensitivities of myoglobin, creatine kinase MB mass, creatine kinase isoform ratios, and cardiac troponins I and T for acute myocardial infarction. ClinChem. 995;4: Perry SV. The regulation of contractile activity in muscle. BiochemSoc Trans. 979;7: Bucher EA, Maisonpierre PC, Konieczny SC, et al. Expression of the troponin complex genes: transcriptional coactivation during myoblast differentiation and independent control in heart and skeletal muscles. Mo! Cell Biol. 988;8: Bodor GS, Poterfield D, Voss EM, et al. Cardiac troponin I is not expressed in fetal and healthy or diseased adult human skeletal muscle tissue. Clin Chem. 995;6:70-75.

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