Additional use of immunostaining for active caspase 3 and cleaved actin and PARP fragments to detect apoptosis in patients with chronic heart failure.
BACKGROUND
Apoptosis might play a role in the pathophysiology of chronic heart failure (CHF). Detection of apoptosis in tissue mostly relies on the terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) method. Recent studies showed, however, that TUNEL-positive cardiomyocytes were not in apoptosis. Therefore, additional methods are warranted. In this study, we tested the specificity of the TUNEL method by additional immunostaining for enzymes and cleaved proteins that are considered specific for apoptosis.


RESULTS
We tested 25 myocardial tissue sections from 16 patients with severe CHF (caused by either idiopathic or ischemic cardiomyopathy) for the existence of apoptosis. We compared TUNEL with immunostaining for caspase 3, active caspase 3, cleaved actin, and poly (ADP-ribose) polymerase (PARP). TUNEL-positive myocytes were seen in 68% of the specimens. Immunostaining in myocytes for active caspase 3 and caspase 3 was positive in only a few patients, whereas staining for cleaved actin and PARP was negative in all patients.


CONCLUSIONS
We found variable myocyte labeling with TUNEL in the failing myocardium of patients with CHF and only obtained scarce additional immunohistochemical evidence for myocyte apoptosis with immunostaining for active caspase 3 and caspase 3 and no positive myocytes with immunostaining for cleaved actin or PARP fragments. These findings support the notion that TUNEL-positive cardiomyocytes are not conclusive for the existence of apoptosis.
de Boer
R A
RA
Department of Cardiology, Thoraxcenter, University Hospital Groningen, The Netherlands.
van Veldhuisen
D J
DJ
van der Wijk
J
J
Brouwer
R M
RM
de Jonge
N
N
Cole
G M
GM
Suurmeijer
A J
AJ
D000199:Actins / Q000032:analysis*; D000328:Adult; D000368:Aged; D017209:Apoptosis; D016022:Case-Control Studies; D053148:Caspase 3; D020169:Caspases / Q000032:analysis*; D002908:Chronic Disease; D005260:Female; D006333:Heart Failure / Q000145:classification / Q000201:enzymology* / Q000209:etiology / Q000378:metabolism / Q000473:pathology*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods* / Q000592:standards; D020287:In Situ Nick-End Labeling / Q000379:methods / Q000592:standards; D008297:Male; D008875:Middle Aged; D009206:Myocardium / Q000648:ultrastructure*; D009210:Myofibrils / Q000201:enzymology* / Q000473:pathology*; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis*; D012680:Sensitivity and Specificity; D012720:Severity of Illness Index
D000199:Actins; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases