NCAM(CD56) and RUNX1(AML1) are up-regulated in human ischemic cardiomyopathy and a rat model of chronic cardiac ischemia

Abstract

Chronic myocardial ischemia is the leading cause of impaired myocardial contractility and heart failure. To identify differentially expressed genes in human ischemic cardiomyopathy (ICM), we constructed a subtracted cDNA library using specimens of ICM compared to normal human heart. Among 100 randomly sequenced clones, seven sequences represented recently identified candidate genes for differential expression in cardiac hypertrophy. A further clone without a known hypertrophy-association coded for the adhesion molecule NCAM(CD56). RNase protection assay, immunohistochemistry, and Western blotting revealed strong overexpression of NCAM(CD56) in all hearts with ICM (n = 14) compared to normal hearts (n = 8), whereas in congestive cardiomyopathy (CCM) (n = 8), hypertrophic obstructive cardiomyopathy (n = 2), myocarditis (n = 4), and sarcoidosis (n = 2), at most slight overexpression of NCAM(CD56) was observed. NCAM(CD56) overexpression abnormally involved the whole cell membrane and the cytoplasma of cardiomyocytes only inside and adjacent to ischemia-induced cardiac scars. Normal or hypertrophic fibers at a distance from ischemic scars were devoid of NCAM overexpression. Identical alterations were observed in an experimental rat ICM model, but not in normal nor in spontaneously hypertensive rat hearts. In search of NCAM(CD56)-related transcription factors we found RUNX1(AML1) up-regulation in ICM and detected RUNX1(AML1) binding within the NCAM(CD56) promoter by electromobility shift assay. We concluded that strong overexpression of NCAM(CD56) and RUNX1(AML1) is a constant and characteristic feature of cardiomyocytes within or adjacent to scars in ICM.

Figure 1.

RNase Protection Assay (a) and Western blot (b) of normal human control hearts (lanes 1 to 4 in a and b) and ICM hearts (a, lanes 5 to 10; b, lanes 5 to 8). Strong overexpression of NCAM(CD56) message and protein in ICM. Slight but significant NCAM(CD56) mRNA and protein overexpression in CCM (a, lanes 11 to 14; b, lanes 9 to 10). Equal loading of mRNA or protein, respectively, per lane checked by GAPDH (a) and muscle-specific actin Western blot (b). up, unprotected radioactive probe; p, protected radioactive probe.

Figure 2.

NCAM(CD56) expression in normal human heart at the disci intercalcares in longitudinal (a) and cross-sections (b). Overexpression and abnormal membrane and cytoplasmatic staining in ICM in longitudinal (c) and cross-sections (d). Restriction of NCAM(CD56) overexpression to cardiomyocytes inside and in contact with a scar (arrows) in ICM (e). Focal and slightly increased expression of NCAM(CD56) in the cytoplasma of cardiomyocytes (f) as well as in cardiomyocytes inside and adjacent to scars in longitudinal (g) and cross-sections (h) in CCM. Immunoperoxidase, ×200.

Figure 3.

Immunohistochemical staining for NCAM(CD56) (a) and cytokeratin 7 (b) of a breast carcinoma infiltrating the musculus pectoralis major. Muscle fibers surrounded by or directly adjacent to carcinoma cells overexpress NCAM(CD56) protein. No NCAM(CD56) overexpression in muscle fibers at a distance from the carcinoma cells.

Figure 4.

Immunohistochemical detection of NCAM(CD56) at the disci intercalcares in a normal rat heart (a). NCAM(CD56) overexpression on the whole cell membrane and in the cytoplasm of cardiomyocytes adjacent to a cardiac scar in rat heart with experimental ICM (b). Normal cardiac NCAM(CD56) expression in a spontaneously hypertensive rat (c). Immunoperoxidase, ×200.

Figure 5.

Various cardiac pathologies and associated NCAM(CD56) expression. Autopsy samples of heart tissue from patients suffering from sarcoidosis (a and b), eosinophilic myocarditis (c and d), acute heart infarction adjacent to an old ischemic scar (e and f) and HOCM (g and h). Either very slight or no up-regulation of NCAM(CD56) is seen on the cardiomyocytes adjacent to or within the inflammatory infiltrate in sarcoidosis, eosinophilic myocarditis, and HOCM. In a case of ICM (case 313/95, e and f) clear overexpression of NCAM(CD56) is detected in the cytoplasma and on the whole cell membrane of viable cardiomyocates within or adjacent to scars (lower right half) whereas no NCAM(CD56) expression is detectable on non-viable, hypereosinophilic cardiomyocytes in a region of acute cardiac infarct (upper left half). HE, Immunoperoxidase ×200.

Figure 6.

Western blots of normal human hearts (lanes 1 to 4) and ICM (lanes 5 to 8) with antibodies against ICAM-1 (CD54), PECAM (CD31), and BL-CAM (CD22). Slight increase only of PECAM in ICM compared to control.

Figure 7.

Detection of NCAM(CD56)-related transcription factors NF-kappa B, HOXD9, PAX (a), and RUNX1(AML1) (b and c) in normal and ICM hearts. a: Similar amounts of NF-kappa B p65 and p50, HOXD9, and PAX 2/5/8 in normal (lanes 1 to 4) and ICM hearts (lanes 5 to 8) (Western blots). b: Overexpression of RUNX1(AML1) mRNA in ICM (lanes 3 to 6) compared to normal human hearts (lanes 7 to 10) and slight overexpression in CCM (lanes 11 to 12). Jurkat-derived (lane 1) and TE671 rhabdomyosarcoma-derived (lane 2) control mRNAs (RNase protection assays). c: Overexpression of the 52-kd RUNX1(AML1) isoform in ICM (lanes 6 to 9) compared to normal human hearts (lanes 2 to 5) and again either very slight or no overexpression in CCM (lanes 10 to 11) (Western blots). Jurkat cell extract (lane 1, positive control). Strong expression of an additional 38-kd band in all samples, with down-regulation in ICM (lanes 6 to 9) and CCM (lanes 10 to 11). up, unprotected radioactive probe; p, protected radioactive probe

Figure 8.

Detection of specific RUNX1(AML1) protein binding within the human NCAM(CD56) promoter by electromobility shift assay (EMSA). Radioactive DNA-protein complex formation by mixing RUNX1(AML1) protein-expressing Jurkat cell extract with [γ-³²P]ATP radioendlabeled WT NCAM(CD56) consensus probe (lanes 1, 4, and 5, arrow) and as control with [γ-³²P]ATP radioendlabeled WT GMCSF consensus probe (lanes 11, 12, 14 and 15, arrow) but not with [γ³²P]ATP radioendlabeled mutant (mt) NCAM(CD56) probe (lanes 6 to 10). Supershifts of the specific complexes by adding anti-RUNX1(AML1) mAb (lanes 5 and 15, arrowheads). No complex formation between the anti-RUNX1(AML1) antibody and radioendlabeled WT NCAM(CD56) (lane 16) or WT GMCSF (lane 17) consensus probes in the absence of RUNX1(AML1) (ie, Jurkat nuclear extract).