Pacing-induced heart failure in dogs enhances the expression of p53 and p53-dependent genes in ventricular myocytes.
BACKGROUND
Rapid ventricular pacing in dogs is characterized by a dilated myopathy in which myocyte cell death by apoptosis may play a significant role in the impairment of cardiac pump function. However, the molecular mechanisms implicated in the modulation of programmed cell death under this setting remain to be identified. Moreover, questions have been raised on the specificity and sensitivity of the histochemical detection of DNA strand breaks in nuclei by the terminal deoxynucleotidyl transferase (TdT) reaction.


RESULTS
Changes in the expression of Bcl-2 and Bax and their transcriptional regulator, p53, were determined by Western blot analysis in myocytes isolated from dogs affected by pacing-induced heart failure. A mobility shift assay for p53 binding activity was also performed. In addition, apoptosis was measured by confocal microscopy, which allowed the simultaneous detection of chromatin alterations and DNA damage. p53 DNA binding activity to the bax promoter was increased in nuclear extracts from myocytes obtained from failing hearts, and this response was associated with enhanced expression of Bax protein, 52%, and attenuation of Bcl-2, -92%. Immunolabeling of p53 in myocyte nuclei, measured by confocal microscopy, was 100% higher in cells from paced hearts. The combination of the TdT assay and confocal microscopy demonstrated that 20 myocyte nuclei per 10(6) were undergoing apoptosis in control myocardium and 4000 per l0(6) after pacing. Moreover, DNA laddering was shown in myocytes by agarose gel electrophoresis of DNA fragments.


CONCLUSIONS
The activation of p53 and p53-dependent genes may be critical in the modulation of myocyte apoptosis in pacing-induced heart failure.
Leri
A
A
Department of Medicine, New York Medical College, Valhalla 10595, USA.
Liu
Y
Y
Malhotra
A
A
Li
Q
Q
Stiegler
P
P
Claudio
P P
PP
Giordano
A
A
Kajstura
J
J
Hintze
T H
TH
Anversa
P
P
D000818:Animals; D015153:Blotting, Western; D002304:Cardiac Pacing, Artificial; D002311:Cardiomyopathy, Dilated / Q000378:metabolism* / Q000503:physiopathology; D017931:DNA Primers; D004285:Dogs; D004587:Electrophoresis, Agar Gel; D005786:Gene Expression Regulation; D019254:Genes, bcl-2; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism*; D066298:In Vitro Techniques; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis*; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis* / Q000378:metabolism; D016276:Ventricular Function; D051028:bcl-2-Associated X Protein
D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein