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.