Author + information
- Mariela Agosto, MD,
- Michael Azrin, MD,
- Kanwar Singh, MD,
- Allan S. Jaffe, MD and
- Bruce T. Liang, MD⁎ ()
- ↵⁎Calhoun Cardiovascular Center, University of Connecticut School of Medicine, 263 Farmington Avenue, MC3946, Farmington, Connecticut 06032
To the Editor:
Infarct size is a major determinant of mortality after acute myocardial infarction (AMI) (1). Although timely reperfusion of the ischemic myocardium in patients with ST-segment elevation myocardial infarction (STEMI) is the most effective way to limit infarct size, reperfusion itself may cause damage, which is known as reperfusion injury (2). Animal data suggest that much of the apoptosis that occurs during occlusion is augmented during reperfusion. Although animal studies and, to a lesser extent, human investigations have suggested the presence of cardiac myocyte apoptosis after AMI, more direct evidence of apoptosis in humans during the acute phase of myocardial infarction (MI) is lacking.
In the present study, we used an enzyme-linked immunosorbent assay to determine the serum level of a cleaved p17 fragment (p17) of caspase-3, the end effector caspase for apoptosis (3), in patients with STEMI.
We studied 27 consecutive patients undergoing primary percutaneous coronary intervention (PCI) for acute STEMI with a door-to-balloon time <90 min from December 2007 to January 2010 at the University of Connecticut Health Center. Informed consent and protocol were approved by institutional review board at our institution.
We measured caspase-3 with a commercial sandwich ELISA assay (PathScan Cleaved Caspase-3 [Asp175] Sandwich ELISA Kit, Cell Signaling Technology, Beverly, Massachusetts). The amount of p17 was determined by analysis of optical density at 450 nm (OD450) using a Multi-Detection Microplate Reader (Biotek, Model Synergy 2, Winooski, Vermont). The p17 peptide levels were linearly related to the amount of apoptotic HeLa cell lysates after staurosporine (1 μM) exposure for 3 h at 37°C; serum p17 OD450 was in the linear range.
The p17 levels were measured at least twice 8 h apart during the initial 24 h, and the peak level was determined as the higher of the AMI values. A subsequent determination was made 88 ± 29.3 days (± SE) after the AMI and is considered the post-MI value. Cardiac troponin I (cTnI) was determined by the Access AccuTnI assay (Beckman Coulter, Brea, California) and creatine kinase (CK)-MB determined by the Access CK-MB assay using Beckman DXI 800 (Beckman Coulter) every 8 h for 24 to 36 h with the 99th percentile value as the upper limit.
Given that cTnI, CK, CK-MB, and p17 values are not normally distributed, nonparametric testing was used with a 2-sided p value <0.05 as significant.
All patients had occluded infarct-related arteries (Thrombolysis In Myocardial Infarction flow grade 0 or 1). Post-PCI Thrombolysis In Myocardial Infarction flow grade was 3 in 89% of patients and grade 2 for the remaining patients. Age was 56.9 ± 10.6 years (± SD) with 93% males. Left ventricular ejection fraction, by left ventriculogram (n = 23) or echocardiogram (n = 4), was 50.0 ± 8.1% (± SD).
The cleaved caspase-3 p17 peptide was shown to escape apoptotic HeLa cells by being detectable in extracellular medium after induction of apoptosis, supporting the concept that p17 peptide can escape into circulation from cells in patients. The correlation of p17 values in a group of 50 healthy control subjects repeated on 2 consecutive days was 0.522, p = 0.0001 (Spearman, median of 0.037 with interquartile range [IQR] 0.030 to 0.043 on day 1 compared with a median of 0.039 with IQR 0.033 to 0.046 on day 2).
The peak serum p17 peptide level during AMI was higher than the late post-MI serum level in 21 subjects in whom samples were available during both phases (peak median OD450 of 0.080 with IQR 0.050 to 0.091 vs. post-median OD450 of 0.052 with IQR: 0.034 to 0.072 respectively, p = 0.0016, Wilcoxon signed-rank test) (Fig. 1). Compared with healthy subjects, the peak AMI p17 level (median of 0.080 with IQR: 0.050 to 0.091) was nearly 4-fold higher than that in healthy subjects (median of 0.041 with IQR: 0.034 to 0.050, n = 167 subjects, Mann-Whitney U test, p < 0.0001). The late post-MI p17 peptide level (median of 0.052 with IQR: 0.034 to 0.073) was also higher than that of healthy subjects (p = 0.016), consistent with persistent apoptosis after AMI.
In all subjects, the peak p17 peptide level occurred after PCI and was positively correlated with the peak cTnI (r = 0.48, p = 0.011) and peak CK-MB (r = 0.56, p = 0.002, Spearman for all) levels. The correlations were not tight, suggesting that both necrosis and apoptosis play a role in determining infarct size. The use of this novel assay for the first time sets the stage to investigate this critical balance. The rise and fall of p17 peptide and its correlation with peak cTnI and CK-MB suggest that serum p17 likely came from injured myocardium. A calpain (4) and stretch-mediated (5) myocyte apoptosis may have also contributed to the serum p17 level.
Limitations include the lack of concurrent myocyte evidence of apoptosis and unknown p17 release kinetics. Designing therapies to inhibit apoptosis may provide benefit to STEMI patients.
Please note: This work was supported by a grant from the Department of Public Health, State of Connecticut (2#010-0083). Dr. Jaffe is a consultant for Beckman, Siemens, Abbott, Roche, Inverness, and Ortho. All other authors have reported that they have no relationships to disclose. The authors thank Jayne Schumacher for assistance in patient enrollment.
- American College of Cardiology Foundation
- Feng J.,
- Schaus B.J.,
- Fallavollita J.A.,
- Lee T.-C.,
- Canty J.M.
- Anversa P.,
- Olivetti G.,
- Meggs L.G.,
- Sonnenblick E.H.,
- Capasso J.M.