Activation of the poly(ADP-ribose) polymerase pathway in human heart failure

Abstract

Poly(ADP-ribose) polymerase (PARP) activation has been implicated in the pathogenesis of acute and chronic myocardial dysfunction and heart failure. The goal of the present study was to investigate PARP activation in human heart failure, and to correlate PARP activation with various indices of apoptosis and oxidative and nitrosative stress in healthy (donor) and failing (NYHA class III-IV) human heart tissue samples. Higher levels of oxidized protein end-products were found in failing hearts compared with donor heart samples. On the other hand, no differences in tyrosine nitration (a marker of peroxynitrite generation) were detected. Activation of PARP was demonstrated in the failing hearts by an increased abundance of poly-ADP ribosylated proteins. Immunohistochemical analysis revealed that PARP activation was localized to the nucleus of the cardiomyocytes from the failing hearts. The expression of full-length PARP-1 was not significantly different in donor and failing hearts. The expression of caspase-9, in contrast, was significantly higher in the failing than in the donor hearts. Immunohistochemical analysis was used to detect the activation of mitochondrial apoptotic pathways. We found no significant translocation of apoptosis-inducing factor (AIF) into the nucleus. Overall, the current data provide evidence of oxidative stress and PARP activation in human heart failure. Interventional studies with antioxidants or PARP inhibitors are required to define the specific roles of these factors in the pathogenesis of human heart failure.

Figure 1

Detection of oxidized proteins. About 0.3 g (wet weight) of ventricular tissues was homogenized in RIPA buffer; 3 μg protein was used for the experiments. Samples are indicated above the lanes (F, failing; D, donor). The optical densities of oxidized protein–derived bands (the evaluated band is indicated) were corrected to the loading, and mean values and SEM are shown in the bar graph (donor, n = 5; failing, n = 8; P = 0.02).

Figure 2

Detection of protein nitration. Human ventricular tissue homogenates (F, failing; D, donor) were loaded (30 μg of protein in each lane) onto 5% to 20% gradient SDS-polyacrylamide gels and probed with a nitrotyrosine-specific antibody (1:10,000; Calbiochem). It should be noted that the major band (indicated) is recognized by the secondary antibody alone and is therefore not specific to nitrotyrosine. A positive control for nitrotyrosine staining (sample F7 treated with 500 μM peroxynitrite for 5 min) is shown on the right.

Figure 3

Detection of poly(ADP-ribosyl)ation. Human ventricular tissue homogenates (D, donor; F, failing) were loaded onto 5% to 20% gradient SDS-polyacrylamide gels (20 μg of protein in each lane) and probed with a poly-ADP-ribose-specific antibody (1:10,000; Calbiochem). According to the manufacturer’s data sheet, this antibody recognizes poly-ADP-ribosylated proteins specifically and serum albumin nonspecifically (indicated). The most prominent nitrotyrosine-specific band (indicated) was further evaluated for purposes of densitometry. Mean values and SEM are shown in the bar graph (donor, n = 5; failing, n = 8; P = 0.005).

Figure 4

Immunohistochemical analysis of poly-ADP-ribosylation. Poly-ADP-ribosylated proteins (A) or AIF (B) were detected on frozen sections from donor (a and c) and failing (b and d) left ventricular tissue samples with antibodies against poly-ADP-ribose polymers (1:100; Calbiochem) or against AIF (1:100; Chemicon), using the ABC method and DAB as chromogen. Sections were counterstained with hematoxylin. Control sections from donor and failing hearts were simultaneously stained with omission of the primary antibody and no significant staining was observed (data not shown). Three samples were evaluated from both donor and failing hearts and representative pictures are shown. The indicated regions on the pictures recorded with low magnification (squares in pictures a, b, c, and d) are also shown in higher magnification, labeled by capital letters (A, B, C and D), respectively.

Figure 5

Detection of poly(ADP-ribose) polymerase (PARP-1) expression. Human ventricular tissue samples (F, failing; D, donor) were loaded (15 μg of protein in each lane) onto 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with a PARP-1–specific antibody (1:10,000; Calbiochem) and also stained with Ponceau. In the densitometric analysis, the optical density of the band representing full-length PARP-1 (indicated) was corrected with the loading, and the mean values ± SEM are shown in the bar graph (donor, n = 5; failing, n = 7; P = 0.33).

Figure 6

Detection of caspase-9 expression. Human ventricular tissue samples (F, failing; D, donor) were loaded (15 μg of protein in each lane) onto 10% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with a caspase-9–specific antibody (1:1000; Cell Signaling) and also stained with Ponceau. In the quantification, the optical density of the band denoting the full-length caspase-9 (procaspase-9, indicated) was corrected with the loading, and the mean values ± SEM are shown on the bar graph (donor, n = 5; failing, n = 8; P = 0.009).