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Proliferating coll nuclear antigen (PCNA) myocyte expression and histoputhologic features related to its occurrence were investigated in normal and diseased hearts of adult humans using both inimunohistochemical and Western blotting techniques. Ki67 Western blotting was also performed in the same samples used for PCNA blotting. Two hundred seventy-one endomyocardial biopsies, and 15 adult, 1 embryonic and 2 fetal hearts were studied. The biopsies were from normal donor hearts (n = 71), patients with cardiomyopathy and myocarditis (n = 64), and patients with transplantation with (n = 106) and without (n = 30) acute rejection of any grade. The 1S hearts were from 1 heart donor, and from patients with cardiomyopathy (n = S), valvular heart disease (n = 2), ischemic heart disease (n = 4), amyloidosis (n = 1) and transplantation with acuto rejection (n = 2). The PCNA labeling index was plotted against myocyte hypertrophy, inflammatory infiltrates and binucleation index. The PCNA labeling index ranged from 2 to 9% in embryonic and fetal hearts. PCNA was expressed by 1 to 2% of myocyte nuclei in 12% of normal heart biopsies, 1 to 5% of myocyte nuclei in 28% of cardiomyopathy and myocarditis biopsies, and by up to 8% of myocyte nuclei in 53% of biopsies of patients with transplantation, independently of the presence and degree of acute rejection. In the latter biopsies and in myocarditis, some inflammatory cells also showed PCNA expression. PCNA positive myocytes were both mono- and binucleated, and there was no correlation between binucleation and PCNA labeling indexes. Ki67 and PCNA blotting confirmed immunohistochemical results. The results show that few myocytes express PCNA in normal hearts. This expression is only detectable by immunohistochemical stains. The percentage of PCNA expressing myocytes increases in diseased hearts of any origin, so that it becomes detectable by both immunohistochemical and Western blotting techniques. Data from transplanted heart biopsies confirm that as in myocarditis, immunologic-inflammatory stimuli are likely powerful triggers of PCNA expression. Therefore, cardiac myocytes of adult humans can express proliferating cell markers, and this expression increases in hearts with hypertrophy or inflammatory reaction. (Am J Cardiol 1993;72:608-614) Embryonic and fetal hearts grow through proliferation of differentiated cardiac myocytes. A few days after birth, myocardial tissue loses its proliferative 'capacity' and soon becomes a 'non-dividing' tissue.[1,2] Growth of myocytes in neonatal rats can be divided into 3 different phases: hyperplastic, transitional and hypertrophic. Myocyte binucleation is considered an early marker of growth hypertrophy and could be the result of mitosis without cytokinesia.[3] Therefore, deoxyribonucleic acid (DNA) of non-dividing cells is probably renewed by mechanisms other than mitotic cell division. The biological half-life of myocyte DNA is estimated to be approximately 40 days with the labeled thymidine method.[4] Given the absence of mitotic division in the myocardium of adult humans,[1-4] the only methods available for investigating possible DNA renewal are those that study cell cycle phases.[4,5] Both thymidine[4] and bromodeoxiuridine incorporation[5] require in vivo administration, but there are no indications for administration of bromodeoxyuridine in cardiac disorders to increase susceptibility of cells to lethal effects of x-radiation, as in neoplastic diseases.[6] Conversely, immunohistochemical methods that detect cell cycle-related antigens can be safely and usefully employed. Examples include Ki67, which identifies a nuclear antigen expressed in all phases except GO,[7,8] and proliferating cell nuclear antigens (PCNA), which are similarly expressed in late G1, S, G2 and M phases of cell cycles.[9,10] The former can be detected only in frozen tissue, whereas the latter can be studied in formalin-fixed, paraffin-embedded samples, because it reacts with a formalin-resistant epitope of PCNA (cyclin). A recent experimental study performed in rat cardiomyocytes demonstrated PCNA messenger ribonucleic acid but not PCNA protein expression in nondividing adult cardiac muscle cells, suggesting post-transcriptional PCNA regulation.[11] To investigate proliferating cell markers in human adult cardiac myocytes and stimuli influencing their expression, we performed immunohistochemical and Western blotting studies using PCNA and Ki67 antibodies in normal and diseased hearts, including cardiomyopathies, other heart diseases and transplanted hearts. PCNA is a 36 KDa nuclear protein that has been identified as an auxiliary molecule of DNA polymerase [sigma].[9] PCNA labeling index correlates with flow cytometric determination of S phase fraction,[12] tritiated thymidine labeling index,[9,13] bromodeoxyuridine incorporation[13,14] and Ki67 labeling index.[15] METHODS Materials: Two embryonic, 1 fetal and 15 adult hearts, and 271 endomyocardial biopsies were investigated. Seventy-one biopsies were from normal donor hearts (22 female and 49 male; mean age 34.4 [+ or -] 13.5 years, range 14 to 61). The hearts had 129 [+ or -] 49 minutes (range 37 to 260) of ischemia between explantation from donor and transplantation. Sixty-four biopsies were from patients (17 women and 47 men; mean age 45 [+ or -] 12 years, range 20 to 62) with the clinical diagnosis of dilated cardiomyyopathy (n = 58), restrictive cardiomyopathy (n = 3), myocarditis (n = 1), Loeffler disease (n = 1) and hypertrophic cardiomyopathy (n = 1). Large right ventricular samples from normal and diseased hearts were also investigated with immunohistochemical and Western blotting methods (see later) to confirm small biopsy results. Finally, 136 biopsies were from transplanted hearts (Table I). All endomyocardial biopsies were perfomed according to the Stanford technique.[16] Four to 6 adequate samples for each procedure were fixed in 10% buffered formalin (20 minutes), dehydrated in a modified alcohol series (95% alcohol: 15 minutes, and 100% alcohol: 15 minutes) and xylene (15 minutes), and embedded in a single paraffin block.[17] For normal donor and idiopathic dilated cardiomyopathy biopsies, 45 sections were serially cut and consecutively collected on 15 slides, and stained at 3 cutting levels (every 75[mu]m) with hematoxylin-eosin and Masson's trichrome or Movat pentachrome. For biopsies from patients with transplantation, 120 sections were serially cut, consecutively collected on 40 slides and similarly stained at 6 cutting levels. Unstained sections were used for immunohistochemical studies. Hispathologic evaluation of endomyocardial biopsies: Normal donor biopsies were studied using previously reported methods and criteria.[18] Idiopathic dilated cardiomyopathy biopsies were evaluated following histopathologic parameters described previously.[19] Acute cardiac rejection was diagnosed according to Stanford criteria and grading.[20] We only distinguished between focal (type A) and diffuse (type B) moderate rejection, with special reference to extension of myocyte damage and necrosis.[21] In the present study, transplantation biopsies are reported with both original diagnosis and grade, and Working Formulation--International Society of Heart Transplantation new grading for acute rejection.[22] Light microscopy immunohistochemistry: Immunohistochemical staining was performed on sections consecutive to those stained with hematoxylin-eosin, Masson's trichrome and Movat pentachrome. Sections were deparaffinized and brought to Tris-phosphate-buffered saline solution (Tris-buffered saline 0.15 M, pH 7.35). After blocking endogenous peroxidase with 3% [H.sub.2][O.sub.2], and pretreatment with type XXVII protease (Sigma Chemicals, St. Louis) or trypsin, the slides were incubated overnight with the primary antisera. Immunohistochemical staining was performed with the peroxidase-antiperoxidase method[23] and avidin-biotin complex technique.[24] Diaminobenzidine (Sigma) was used as chromogen substrate. Specificity tests, performed by omission of the first layer, produced negative results. PCNA monoclonal antibodies were commercially obtained (Dako A/S, Denmark). The optimal working dilution was 1:300. Titration tests were performed on routinely fixed specimens, including normal (lymphatic and epithelial) and neoplastic (breast carrinomas, central nervous system neoplasms and bowel tumors). Quantitation of binucleated and proliferating cell nuclear antigen-labeled nuclei: The percentages of binucleated myocytes and immunolabeled nuclei were semiquantitatively evaluated in all samples using 25 X objective lens. Binucleation was evaluated in longitudinally cut myocytes. In cases with significant field-to-field variation, the highest and lowest scores were indicated. We used this method because no image analysis system can distinguish between myocyte and endothelial or interstitial cell nuclei. In addition, semiquantitative techniques have been reported to provide reproducible and useful results in other tissues.[12] Immunoblotting: We also used immunoblotting techniques on multiple samples to test the presence and specificity of anti-PCNA antibodies. The samples were from the following heart types: 1 normal donor (24-year-old man who died from cerebral trauma); 5 with idiopathic dilated cardiomyopathy; 4 with coronary artery disease; 2 with valvular heart disease; 1 explanted heart with amyloidosis; and transplanted hearts with acute rejection obtained at either retransplantation (n = 1) or autopsy (n = 1). All but 1 of the samples were from men in the age range of 24 to 58 years (mean 47.5 [+ or -] 10.4). Biopsy samples are usually too small to extract adequate amounts of protein for blotting. Methods were previously described in detail.[25] Polyacrylamide 10% gel electrophoresis was performed according to the Laemmly method.[26] Proteins were transferred to nitrocellulose according to Towbin et al.[27] The presence of Ki67 antigen (Ki67 monoclonal antibody, Dako A/S) was also tested in the samples used for PCNA blotting and for immunohistochemical stains by sodium dodecyl sulphate polyacrylamide gel electrophoresis with a low percentage (5%) acrylamide stab gel system.[28] The membranes were then incubated with the primary antibodies for 1 hour. Bound antibody was targeted with alkaline phosphatase conjugated with goat anti-mouse immunoglobulin G. Finally, bound alkaline phosphatase was visualized following the method of Leary et al.[29] Statistical analysis: To verify the presence of association between categoric variables (Table II), chi-square tests were performed, and C contingency coefficients were computed for the normal heart donor group, as well as for the cardiomyopathy group. The variable 'age' was transformed into 20-year interval classes for testing against other variables. For testing between ordinal variables, Kendall tau coefficients were also computed. RESULTS PCNA-immunolabeled nuclei were sharp and easily identified in both myocyte and interstitial cells. Only myocyte nuclei were considered. Nuclear staining pattern were both diffuse and finely reticular, and exhibited variable intensity. Immunohistochemical results in fetal hearts: Embryonic and fetal hearts had a high PCNA labeling index ranging from 2 to 9% (Figure 1). The highest scores were obtained from the interventricular septum and left ventricular walls. Immunohistochemical results in normal donor biopsies: PCNA labeling was detected in myocyte nuclei of 8 of 71 donor biopsies (11.27%). Few positive endothelial and interstitial mesenchymal cells were observed in 1 case. In the 8 positive cases, the PCNA labeling index ranged from 1 to 2%. Binucleated myocytes were observed in 50 of 71 biopsies (70.42%), and binucleation index ranged from 0 to 3%. Myocyte hypertrophy (transversal 0 >20 [mu]m) was found in 25 biopsies (35.2%), and some fibrosis in 24 (33.8%). No relation was observed between PCNA labeling, and age, myocyte hypertrophy and binucleation (Table II), because PCNA protein expression was detected in both mono--and binucleated myocytes (Figure 2). Immunohistochemical results in biopsies of patients with idiopathic dilated cardiomyopathy: Histopathologic study of the biopsies of patients with idiopathic dilated cardiomyopathy showed features consistent with cardiomyopathy in 31 cases (including 1 arrhythmogenic right ventricular disease), myocyte hypertrophy or interstitial fibrosis, or both, in 13, granulomatous myocarditis in 3, healing myocarditis in 2, isolated myocyte hypertrophy in 7, Loeffler endocardial disease in 1, endomyocardial fibrosis in 1, small vessel disease in 1, cardiac amyloidosis in 1, sparse lymphocytes with no hypertrophy or fibrosis in 1, and histologically normal myocardial tissue in 3. PCNA labeling index showed high field-to-field variation in the percentage of positive cells, ranging from 1 to 4% in positive cases. Binucleation index varied from 0 to 5%. The intensity and pattern of immunoreactivity were similar to those observed in normal donor biopsies, but positive immunoreaction occurred mostly (but not exclusively) in hypertrophic cardiomyopathy and huge, bizarre nuclei of idiopathic dilated cardiomyopathy biopsies (Figure 3). In myocarditis and the Loeffler case, some inflammatory cells had sparse labeled nuclei that were not counted. An association was found between age and binucleation index on the one hand (Kendall tau - 0.245), and between PCNA and binucleation index with a higher contingency coefficient on the other (Table II). Immunohistochemical results in transplanted heart biopsies: In this series, we detected a very high percentage of positive biopsies (53%). Grouping of these biopsies according to presence of acute cardiac rejection and degree of rejection itself revealed no difference between rejection-free biopsies (Figure 4, a, b and c) and those with mild (Figure 4d) or moderate Figure 5, a and b) rejection (Table I). Contrary to idiopathic dilated cardiomyopathy and noninflammatory diseases, and analogous to inflammatory diseases, interstitial inflammatory cells other than myocytes showed definite nuclear immunostaining. PCNA myocyte labeling index was higher in transplanted hearts than normal or idiopathic dilated cardiomyopathy hearts, reaching values as high as 8%. Immunoblotting results: Antibodies to PCNA recognized a 36 KDa-band polypeptide in immunoblotting studies of large samples from hearts with idiopathic dilated cardiomyopathy, ischemic heart disease, valvular heart disease, amyloidosis and acute rejection, whereas no bands were observed in normal heart samples. Negative immunoblotting findings in normal heart samples indicate that the method does not detect the small amounts of PCNA protein clearly shown by immunohistochemical study. Antibodies to Ki67 recognized a 354 KDa polypeptide not only in the same samples expressing PCNA (9 of 14 diseased hearts; 62.3%), but also in 3 additional cases (12 of 14; 85.7%). Comparison between different methods: Immunohistochemical stains detected single PCNA-positive nuclei, whereas blotting did not detect the very low protein amount present in normal hearts. The probability of finding PCNA immunoreactivity increased with sample size, so that PCNA expression was found in 40% of large versus 26% of small idiopathic dilated cardiomyopathy biopsies. In addition, PCNA expression was not influenced by preservation. When using Western blotting techniques on extracts from nonfixed tissue, we found that 40% of idiopathic dilated cardiomyopathy myocardial samples expressed PCNA. Some PCNA derived from myocyte nuclei, because immunohistochemical staining did not show labeling of interstitial or endothelial cell nuclei in idiopathic dilated cardiomyopathy samples. Finally, blotting with Ki67 revealed 80% of idiopathic dilated cardiomyopathy samples to be positive (Table III). The highest Ki67 expression found by blotting in diseased hearts was probably related to its large size and high molecular weight. DISCUSSION This is the first report documenting expression of PCNA protein in normal adult human myocytes. PCNA expression increases in different disorders primarily affecting myocardium, such as idiopathic dilated cardiomyopathy and myocarditis, and in transplanted hearts where myocardial tissue is subjected to chronic immunologic stimuli. This PCNA expression suggests the possibility that periodic DNA renewal occurs in human adult myocytes, and that expression rate can be influenced by hypertrophy and immune- or inflammatory-mediated stimuli. Therefore, although myocardium is a non-dividing tissue, mechanisms other than mitotic cell replication could intervene in DNA renewal in stimulated myocytes. The data provide new insights into myocardial expression of proliferating cell markers. Functionally normal adult myocardium: There is definite evidence that during fetal life cardiac muscle cells synthesize DNA and divide.[1,2,4] Our embryonic and fetal hearts did not show mitosis, but did show PCNA immunoreactivity. This is in accordance with blotting data reported by Marino et al.[11] The percentage of labeled myocytes (labeling index) after 24 hours of ([sup.3]H)thymidine incorporation is inversely related to heart growth, so that the labeling index is at a maximum during early fetal life, progressively reduces after birth and is negligible by the end of the third week.[4] The present results in normal donor biopsies are in accordance with data obtained from experimental animals. We found PCNA-labeled myocyte nuclei in 12% of normal biopsies. In the positive cases, the PCNA labeling index was very low (1 to 2%). Other studies investigating myocardial PCNA expression did not find any staining in adult cardiac myocytes.[10] These different results could be related to technical processing of biopsy samples. Fixation time can greatly alter detection of PCNA immunoreactivity. The short fixation time, together with previously described, modified processing methods[17] used in this study, could explain these differences. We did not find any significant correlation between PCNA protein expression and age, hypertrophy and binucleation index in the normal heart group. Binucleated cardiac myocytes, early indicators of growth hypertrophy,[3] are a frequent finding in normal adult human hearts, representing up to 3% of myocytes. In the present study, PCNA immunolabeling was detected in some binucleated myocytes, whereas it was not found in many other binucleated myocytes. Therefore, binucleation and PCNA protein expression are not necessarily related to each other. The negative results obtained by blotting with PCNA in normal hearts confirms that the amount of PCNA protein is too small to be detected by this technique. The negative results obtained in normal heart samples with Ki67 blotting could have 2 possible interpretations. The first possibility is that Ki67 is not expressed at al, whereas the second is that as with PCNA, the technique is not sufficiently sensitive to detect small amounts of Ki67 possibly expressed in normal hearts. The present PCNA blotting results are in accordance with those reported by Marino et al[11] in their Western blotting study of adult experimental hearts. Conversely, immunohistochemical techniques proved to be more sensitive than did blotting, because they detected PCNA protein expression even in single cells and were also more useful for precisely defining labeled cell phenotype. Accordingly, very low but definite PCNA expression in functionally normal, adult human hearts suggests that there is a low but constant rate of DNA synthesis, indicating the possibility of amitotic DNA renewal. Diseased adult hearts: Percentages of PCNA immunolabeling in this series varied according to pathologic entities, reaching a maximal value in myocarditis and the lowest percentages in cardiomyopathies; in the former, positive myocyte nuclei were found in all biopsies, whereas in the latter, the incidence of positive cases was twice that of normal hearts. The paucity of cases with other, rarer disorders prevents consideration of such disorders. However, there is a trend toward increasing expression of PCNA from normal to diseased hearts with abnormal stimuli, such as inflammation or hypertrophy. Previous studies indicated that as diseased hearts increase in size and age, the ploidy of the cardiac muscle cells increases.[30-32] Those data suggest that some new DNA synthesis occurs in human adult cardiac myocytes, and are in accordance with our finding of PCNA positivity in cases with hypertrophy of any origin and nature (i.e., primary, such as in cardiomyopathy, or secondary, such as that of valvular heart disease). Binucleation correlated with patient age, suggesting that it can be an age-related feature (as can hypertrophy).[18] In addition, in cardiomyopathy hearts, binucleation index correlated with PCNA nuclear immunolabeling, although some positive nuclei were single. This result could be consistent with abnormal DNA renewal or repair[33] in huge nuclei often observed in cardiomyopathies.[19] With respect to normal control cases, PCNA overexpression by myocyte nuclei in the different primary or secondary noninflammatory disorders, as well as Ki67 expression found by blotting, suggests that proliferating cell antigen expression increases in hearts with stimuli of any origin and type. The actual significance of PCNA immunolabeling in myocarditis is not clear. Inflammation could stimulate myocyte DNA renewal, as well as cause cardiac damage needing DNA repair.[33] Accordingly, myocytes could respond to inflammatory stimuli by regenerating their sarcoplasmatic and sarcolemmal components after DNA renewal or repair. In any PM of myocardial inflammatory injury, nuclei are the last cell components to die. Transplanted heart biopsies: The highest percentages of PCNA protein expression, together with the highest PCNA labeling index, were found in biopsies from transplanted hearts. The presence of acute rejection of any degree (mild or moderate) did not influence PCNA expression, because positive nuclei were observed in biopsies both with and without acute rejection. The PCNA labeling index was also similar in rejection and nonrejection groups, suggesting that permanent immunologic stimuli, rather than grade, probably influence expression of DNA renewal markers. The presence of labeled nuclei in the interstitial inflammatory or mesenchymal cells was not related to myocyte nuclei labeling, because the latter was found in areas both with and without infiltrates. The positive blotting results for both PCNA and Ki67 in transplanted hearts with acute rejection could be due to both myocytes and inflammatory or interstitial positive cells detected by immunohistochemistry. However, immunohistochemical study showed that at least part of immunoreactivity is definitely due to myocyte nuclei PCNA expression. The high expression of cell proliferation markers in denervated, transplanted hearts could be positive for myocardial well-being. However, we are still far from understanding the mechanisms regulating myocardial cell replication or renewal. The very recent demonstration that PCNA is needed for DNA excision repair[33] suggests an alternative explanation for PCNA expression in normal and diseased myocardium in humans. In conclusion, the unexpected expression of cell proliferation markers such as PCNA and Ki67 in diseased human adult hearts suggests that myocardium may increase its DNA renewal or repair rate as a result of inflammatory or immunologic injuries and of stimuli triggering primary or secondary hypertrophy. Aknowledgment: We thank Emanuele Porcu for technical assistance, and Linda D'Arrigo for English revision. [1.] Peterson RO, Baserga R. Nucleic acid and protein synthesis in cardia muscle of growing and adult mice. Exp Cell Res 1965;40:340-352. [2.] Rumayantsev PP. Interrelations of the proliferation and differentiation processes during cardiac myogenesis and regeneration. Int Rev Cytol 1977;51:187-273. [3.] Clubb FJ, Bishop SP. Formation of binucleated myocardial cells in the neonatal rat. Lab Invest 1984;50:571-577. [4.] Pelc SR. Labelling of DNA and cell division in so called non-dividing tissues. J Cell Biol 1964;22:21-28. [5.] Sugihara H, Hattori T, Fukuda M. Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues. Histochemistry 1986;85:193-195. [6.] Kriss JP, Maruyama Y, Tung LA, Bond SB, Revesz L. The fate of 5-bromodeoxyuridine, 5-bromodeoxycytidine and 5-iododeoxycytidine in man. Cancer Res 1963;23:260-268. [7.] Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H. Cell cycle analysis of a cell proliferation-associated human nuclear antigen defined by the monoclonal antibody Ki-67. J Immunol 1984;133:1710-1715. [8.] Brown DC, Gatter KC. Monoclonal antibody Ki-67: its use in histopathology. Histopathology 1990;17:489-503. [9.] Mathews MB, Bernstein RM, Franza BR Jr, Garrels JI. Identity of the proliferating cell nuclear antigen and cyclin. Nature 1984;309:374-376. [10.] Hall PA, Levison DA, Woods AL, Yu CCW, Kellock DB, Watkins JA, Barnes DM, Gillett CE, Camplejohn R, Dover R, Waseem NH, Lane DP. Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms. J Pathol 1990; 162:285-294. [11.] Marino TA, Haldar S, Williamson EC, Beaverson K, Walter RA, Marino DR, Beatty C, Lipson KE. Proliferating cell nuclear antigen in developing and adult rat cardiac muscle cells. Circ Res 1991;69:1353-1360. [12.] Garcia RL, Coltrera MD, Gown AM. Analysis of proliferative grade using anti PCNA/cyclin monoclonal antibodies in fixed, embedded tissue. Comparison with flow cytometric analysis. Am J Pathol 1989;134:733-739. [13.] Buttersby S, Andersen TJ. Correlation of proliferative activity in breast tissue using PCNA/cyclin. Hum Pathol 1990;21:781. [14.] Coltrera MC, Gown AM. PCNA/cyclin expression and BrdU uptake define different cell subpopulation in different cell lines. J Histochem Cytochem 1991; 39:23-30. [15.] Levison DA, Hall PA, Woods AL, Yu C, Barnes DM, Wasseem N, Lane DP. Evaluation of PCNA (proliferating cell nuclear antigen) immunostaining as a marker of cell proliferation in formalin-fixed paraffin-embedded tissues (abstr). J Pathol 1990;161:341. [16.] Caves PK, Stinson EB, Billingham ME, Shumway NE. Percutaneous transvenous endomyocardial biopsy in human heart recipients. Ann Thorac Surg 1973; 16:325-336. [17.] Arbustini E, Grasso M, Diegoli M, Bramerio M, Scotti Foglieni A, Albertario M, Martinelli L, Gavazzi A, Goggi C, Campana C, Vigano M. Expression of tumor necrosis factor in human acute cardiac rejection. An immunohistochemical and immunoblotting study. Am J Pathol 1991;139:707-715. [18.] Arbustini E, Gavazzi A, Pozzi R, Pucci A, Grasso M, Diegoli M, Campana C, Graziano G, Martinelli L, Goggi C, Montemartini C, Vigano M. Endomyocardial biopsy of normal heart before cardiac transplantation. A morphologic and morphometric study in 97 cases. Am J Cardiovasc Pathol 1992;4:1-8. [19.] Arbustini E, Gavazzi A, Pozzi R, Grasso M, Pucci A, Campana C, Graziano G, Martinetti M, Cuccia MC, Martinelli L, Salvaneschi L, Montemartini C, Vigano M. The morphologic spectrum of dilated cardiomyopathy and its relation to immune-response genes. Am J Cardiol 1989;64:991-995. [20.] Billingham Me. Diagnosis of cardiac rejection by endomyocardial biopsy. Heart Transplant 1981;1:25-29. [21.] Arbustini E, Grasso M, Diegoli M, Gavazzi A, Campana C, Martinelli L, Goggi C, Pucci A, Grossi P, Ippoliti C, Vigano M. La biopsia endomiocardica nel paziente cardiotrapiantato: stato dell'arte. G Ital Cardiol 1991;21:1107-1123. [22.] Billingham ME, Cary NRB, Hammond ME, Kemnitz J, Marboe C, McA]lister HA, Snovar DC, Winters GL, Zerbe A. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: heart rejection study group. J Heart Lung Transplant 1990;9:587-593. [23.] Stemberger LA. The unlabelled antibody enzyme peroxidase-antiperoxidase (PAP) method. In: Stemberger LA, ed. Immunocytochemistry. New York: John Wiley, 1979:104-169. [24.] Hsu SM, Rainel L, Fanger H. Use of avidin-biotin peroxidase complex (ABC) in immunoperoxidase techniques. J Histochem Cytochem 1981;29:577-580. [25.] Arbustini E, Pucci A, Grasso M, Diegoli M, Pozzi R, Gavazzi A, Graziano G, Campana C, Goggi C, Martinelli L, Silini E, Specchia G, Vigano M, Solcia E. Expression of natriuretic peptide in ventricular myocardium of failing human hearts and its correlation with the severity of clinical and hemodynamic impairment. Am J Cardiol 1990;66:973-980. [26.] Laemmly UK. Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature 1970;227:680-685. [27.] Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 1979;76:4350-4354. [28.] Gerdes J, Li L, Schlueter C, Duchrow M, Wohlenberg C, Gerlach C, Stahmer I, Kloth S, Brandt E, Flad HD. Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67. Am J Pathol 1991;138:867-873. [29.] Leary JJ, Brigatti DJ, Ward DC. Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized tp DNA or RNA immobilized on nitrocellulose: bioblots. Proc Natl Acad Sci U S A 1983;80:4045-4049. [30.] Anversa P, Olivetti G, Loud AV. Morphometric study of early postnatal development in the left and right ventricular myocardium of the rat. Circ Res 1980;46:495-502. [31.] Brodsky WJ, Arefyeva AM, Uryvaeva IV. Mitotic polyploidization of mouse heart myocytes during the first postnatal week. Cell Tissue Res 1980;210:133-144. [32.] Sandritter W, Scomazzoni G. Deoxyribonucleic acid content (Feulgen photometry) and dry weight (interference microscopy) of normnal and hypertrophic heart muscle fibres. Nature 1984;202:100-101. [33.] Shivij MKK, Kenny MK, Wood RD. Proliferating cell nuclear antigen is required for DNA excision repair. Cell 1992;69:367-374. From the Pathology, Cardiac Surgery, and Cardiology Departments, Istituto di Ricovero e Cura a Carattere Scientifico, IRCCS, Policlinico San Matteo, Universita di Pavia, Pavia, Italy. This study was supported by grants 'Trapianto Cardiaco' and 'Trapianto Cuore-Polmoni' from Health Ministry to IRCCS, Policlinico San Matteo, Pavia-Ricerche Finalizzate 1989-1990. Manuscript received October 23, 1992; revised manuscript received March 8, 1993, and accepted March 19. Address for reprints: Eloisa Arbustini, MD, Dipartimento di Patologia Umana ed Ereditaria, Sezione di Anatomia Patologica, Via Forlanini 14, 27100 Pavia, Italy.

Low doses of acetylcholine induce 'endothellum-dependent' dilatation in normal coronary arteries and constriction of diseased vessels. This study investigated morpholoigic changes induced by perfusion of normal and diseased coronary arteries with low and high doses of acutylchollne. Vessels were excised from a series of beating hearts explanted at transplantation for idiopathic dilated cardliomyopathy and coronary artery dim. Coronary arteries from other explanted hearts, perfused with saline solution under similar conditions were taken as control. Samples were studied using conventional histopathologic and immuno-histochemical methods. Coronary arteries were grouped according to presence or absence of his tologically detectable structural modifications of any type and extent. Low doses of acetylcholine Induced changes In all but 1 structurally diseased coronary artery, whereas no change was induced in any but 1 histologically normal coronary artery. High doses of acetylcholine induced contraction changes In both normal diseased vessels. Changes observed in the wall of the contracted vessels were: (1) endothelial cell contraction with protruding nuclei and detachment of their intercellular junctions with exposure of subject collaget to flow, (2) contraction of plaque smooth muscle cells, (3) formation of cushions protruding Into vessel lumens causing blunt microchannels. Contraction in both intimal and plaque culls occurred even in diseased vessel segments with intimal denudation. These effects seemed to be dose-dependent in structurally normal vessels because low doses of acetyicholine did not produce any morphologically detectable changes in histologically normal coronary arteries, while low domes of acetyicholine induced similar reactions in vessels affected by both atherosclerosis and subintimal flbrocellular thickening. (Am J Cardiol 1993;71:1382-1390)

A polymerase chain reaction (PCR) amplification assay was developed to detect Coxsackievirus B3 ribonucleic acid (RNA) in blood and myocardial tissue of explanted hearts from 40 patients who underwent cardiac transplantation and in 1 normal heart. Twenty-one patients were affected duration dilated cardiomyopathy of different duration and 19 by coronary artery disease. Coxsackievirus B3 in vitro infected Vero cells and cells infected by related human enteroviruses (Coxsackievirus B2, B4, and poliovirus 1) were used as reaction controls. PCR was performed using 4 pairs of primers homologous to Coxsackievirus B3 sequences. Three sets were located in regions of the genome conserved at nucleotide level between several entervirus species (replicase gene, 5' noncoding region), while one was located in a Coxsackievirus B3-specific region (VP1 gene). Total RNA was prepared by acid guanidinium isothyocyanate extraction from tissue stored frozen at -80 [degrees] C. One microgram of total RNA was retrotranscribed with either antisense primer or with random hexanucleotide primers and then subjected to 40 cycles of amplification. PCR products were separated byd electrophoresis on a 10% polyacrilamide gel, electrotransferred to a nylon membrane and then hybridized to oligonucleotide probes specific for the coxsackievirus B3 genome radiolabeled with radioactive isotope of phosphorous. All pairs of primers yielded specific amplification products when tested on Coxsackievirus B3-infected Vero cells, with a sensitivity of 1 infected cell out of [10.sup.5] to [10.sup.6] cells starting from 1 [mu]g total RNA. Primer sets for regions of Coxsackievirus B3 genome highly conserved between related enteroviral species gave positive amplification also when challenged with RNA from cells infected by Coxsackievirus B2, B4 and poliovirus 1. The VP1 gene primer set produced positive amplification only with RNA of Coxsackievirus B3-infected cells. Coxsackievirus B3-specific amplification products were distinguished from those of related enteroviruses by hybridization with specific oligoprobes. However, Coxsackievirus B2, B4 and poliovirus 1-specific PCR products showed positive hybridization if probed with Coxsackievirus B3 genomic probes. All total RNAs from blood and myocardial samples examined by our PCR assay failed to reveal any amplification product that could be related to Coxsackievirus B3 or to enteroviruses in general, after gel electrophoresis and low stringency Southern blot hybridization with the Coxsackievirus B3 specific oligoprobes or the Coxsackievirus B3 genomic probes. The negative results obtained in our series question the hypothesized widespread persistence of enteroviral RNA in hearts with idiopathic dilated cardiomyopathy.

Ischemic syndromes are a collection of disorders in which the heart muscle does not receive an adequate supply of blood. Atherosclerosis, in which the arteries of the heart are partially blocked by fat-laden lesions, is very common in ischemic heart disease, but it is also common among people without ischemic heart disease. Ischemic heart disease may be stable or unstable. An example of a stable ischemic condition is stable angina, in which drugs designed to relax the blood vessels and improve blood flow often control the problem. Unstable ischemic syndromes include unstable angina and myocardial infarction (heart attack). These unstable conditions appear to be associated with the formation of blood clots, or thrombi, within the arteries of the heart. The formation of these blood clots seems to begin in an atherosclerotic lesion. Since atherosclerosis is so common, it is important to determine why some atherosclerotic conditions are stable and lead to only relatively benign disease while others are unstable and are more likely to result in heart attack or sudden death. To evaluate the distinctions among different patients with atherosclerosis, samples of artery tissue were examined from patients with stable, unstable, and nonischemic disease. These specimens were obtained during autopsy or surgery. It was found that extensive narrowing (stenosis) of the coronary arteries was common; it was present in 90 percent of cases of stable and unstable disease. Furthermore, stenosis was found in about half the patients with nonischemic disease. Researchers observed, however, that the composition of the plaque seemed to influence the likelihood of clot formation within the artery. Thrombi were more likely to be associated with atherosclerotic lesions that were mostly fatty pulp, that is, pultaceous. In contrast, thrombi were less likely to be associated with atherosclerotic lesions that were mostly fibrous. Since a primary step in the development of a thrombus is the adherence of platelets (cells involved in clotting) to the surface of the atherosclerotic lesion, it would seem that the characteristics of this surface play a major role in the progression of thrombosis. (Consumer Summary produced by Reliance Medical Information, Inc.)

Atrial natriuretic peptide (ANP) is a substance secreted by the atrium of the heart in response to stretching of cardiac tissue (such as occurs during hypertensive episodes or congestive heart failure). It causes excretion of sodium in the urine, dilation of the blood vessels, and other physiological responses that tend to reduce the workload of the heart. Human atrial myocytes (muscle cells) have been shown to contain ANP. In heart failure, ventricular myocytes also appear to contain and secrete ANP. This results from an induction (turning on) of the normally inactive ventricular ANP gene. To investigate the factors involved in the re-expression of the ANP gene in failing human hearts, a microscopic study was undertaken to analyze muscle tissue from 87 healthy hearts, one heart from a patient with hepatitis B, 151 hearts from patients with dilated cardiomyopathy (a type of heart disease), 26 explanted hearts with dilated cardiomyopathy, and 31 explanted hearts with ischemic heart disease (cardiac damage resulting from decreased blood flow). Hearts were treated with specific stains to detect the presence of ANP. The ventricles of normal hearts were completely devoid of ANP. Ventricular ANP was found in 30 to 60 percent of the ventricles of the failing hearts, and the presence and extent of ventricular ANP was not related to the type of heart disease. However, there was a correlation between the amount of ventricular ANP and both the severity of the disease symptoms and several physiological indices of cardiovascular dysfunction. (Consumer Summary produced by Reliance Medical Information, Inc.)

Endomyocardial biopsies from 174 patients with dilated cardiomyopathy (DC) were examined. Eight patients with histologically proven myocarditis were excluded from the study. A peculiar pattern of oversized and bizarre nuclei was observed in only some of the remaining patients. Two groups were identified: those with and without this feature (groups A and B, respectively). Myocyte width, nuclear diameter and nuclear/sarcoplasmic ratio were significantly higher in group A. The mean respective values were 36 [+ or -] 5 [mu], 14 [+ or -] 3 [mu] and 0.41 [+ or -] 0.08 for group A versus 20 [+ or -] [mu], 7 [+ or -] 2 [mu] and 0.37 [+ or -] 0.08 for group B. interstitial fibrosis was similarly present in groups A and B. Endocardial thickness was significantly increased in all patients, with group A showing the highest mean value. The morphologic features showed no correlation with the clinical condition of the patients at time of presentation. HLA typing was performed in 50 consecutive patients, 38 from group A and 12 from group B. DR4 and DR5 antigens were significantly more frequent in DC patients than in a normal population control (400 blood donors), while DR3 was less frequent. Group A was more strongly associated with the DR5 antigen than group B (55.3 vs 25.0%, respectively). it was less strongly associated with the DR4 antigen compared with group B (21.5 vs 41.7%, respectively). No difference was observed between the 2 groups concerning negative association with the DR3 antigen. Endemyocardial biopsies from DC patients reveal marked morphologic changes from patient to patient. The histopathologic identification of 2 distinct DC groups with different HLA marker associations suggests that immune-response genetic factors may play a role in the histopathologic expression of the disease. (Am J Cardiol 1989;64:991-995), Dilated cardiomyopathy (DC) is a disease of the heart muscle in which the muscle cells (myocytes) degenerate and fibrous elements invade the tissue causing the wall to thicken. Genetic factors have been suggested as one possible cause. In particular, the association of specific human leukocyte antigens (HLA), molecules present on the surface of blood cells of every individual, with DC has been proposed. Fifty out of 166 patients diagnosed with DC on the basis of cardiac biopsy also provided blood samples for HLA determination. Biopsies were evaluated using the light microscope and measurements were of myocyte width (average width of the heart cell), various cell nuclei dimensions including diameter, extent of fibrosis (or presence of fibrous tissue), thickness of the heart wall, and other related variables. Blood from 400 hundred healthy blood donors was used to determine HLA antigen frequencies in the general population for comparison with the group with DC. The most important finding regarding muscle cells from DC patients was the presence of very large, abnormal cell nuclei. The magnitude of these changes, however, was not correlated with extent of symptoms or duration of the disease. The 60 percent of patients who had this abnormality constituted Group A, while the remainder made up Group B. When DC patients were compared with the population as a whole, they were found to display HLA antigen subtypes DR4 and DR5 more often, with no difference for DR3. Among DC patients as a group, HLA antigen subtype DR4 was more frequent in Group B than Group A; subtype DR5, on the other hand, was more frequent in Group A patients. The lack of correlation between abnormal cells and disease progression argued against interpreting these changes as a pathological response to failure of the heart's contracting mechanism, since they should then be more numerous in advanced disease. The cell nuclei abnormalities resembled changes noted during activation of protein synthetic machinery. Analysis of HLA subtypes showed that a type of cellular damage can be associated with an HLA subtype. This finding is consistent with the hypothesis that DC is one of several diseases caused by the chemical product of an interaction between a virus and the immune system. The hypothesis holds that this product then becomes toxic to the body's cells. While implications of these results for treatment or progression of DC are not clear, clarification of the role of the immune system with respect to this disease could shed light on its etiology. (Consumer Summary produced by Reliance Medical Information, Inc.)

In normal circumstances, the heart muscle contracts in a synchronized, rhythmic fashion while it pumps blood to the body. In some disease conditions, the heartbeat can become irregular (arrhythmic) or the muscular contractions can become desynchronized (fibrillation), leading to a loss of the heart's ability to pump blood effectively. These disorders of rhythmicity are frequently treated with antiarrhythmic medications; amiodarone is a particularly effective example of this class of drug, and is frequently prescribed. However, it is not without side effects, and its accumulation in different tissues has been reported to cause a variety of adverse reactions. In previous studies, chronic amiodarone administration has been reported to cause the appearance of characteristic subcellular organelles in the liver, lymph nodes, blood cells, skin cells, intestinal mucosa, and nervous tissue. Animal studies have shown that amiodarone treatment can result in the appearance of these bodies in cardiac muscle cells, but this has not been reported in human studies. Two cases are described of patients with dilated cardiomyopathy (a condition in which the heart is enlarged) of unknown cause; a 50-year-old man and a 53-year-old woman had been receiving amiodarone to control cardiac arrhythmias for four and two years, respectively. Biopsy specimens taken from the right ventricle were viewed at high magnification using electron microscopy (enlarged 23,000 times). Numerous subcellular organelles seen within the cardiac myocytes (muscle fibers) of these patients appeared to be identical to those reported in other body tissues by investigators studying the effects of long-term amiodarone use. This finding is not common; of over 300 patients with dilated cardiomyopathy who underwent biopsy, 40 percent were receiving chronic amiodarone treatment, and of the 247 biopsy specimens analyzed, only these 2 showed this alteration. The implications of this morphological alteration are not known. (Consumer Summary produced by Reliance Medical Information, Inc.)