Background: Current proteomic technologies have been slow to identify and validate new cardiac biomarkers. A multiplexed proximity extension assay (PEA) that uses nucleotide-labeled antibodies coupled with real-time PCR quantification may offer the advantage of high-throughput and sensitivity for biomarker discovery.
Methods: We applied a multiplexed PEA that uses nucleotide- labeled antibodies to measure approximately 1,000 proteins in samples from individuals undergoing a “planned” myocardial infarction (PMI: alcohol ablation for hypertrophic cardiomyopathy), in which each individual serves as their own biologic control. Serial blood samples were obtained before and at 10 minutes, 1 hour, 4 hours and 24 hrs after PMI. Patients undergoing patent foramen ovale closure served as negative controls. To further assess the generalizability of our findings, we profiled samples from a spontaneous myocardial infarction (SMI) cohort.
Results: Seventy-one proteins were significantly changed after myocardial injury in our PMI cohort (n=10; p < 5.10 E-05, one-way ANOVA repeated measures), after excluding 40 overlapping proteins identified as significantly changed in the PFO negative controls (n=6; p < 4.50E-04). Protein changes included known cardiac markers of injury such as troponin I (TNNI3; peak change: 57 fold increase at 4hrs, p= 3.96E-17) and myoglobin (MB; peak change: 9 fold increase at 1 hr, p= 4.250E-12). Many protein changes detected are novel in the context of myocardial injury, including mitochondrial 2,4-dienoyl-CoA reductase, a beta oxidation enzyme important in fatty acid metabolism (DECR1; peak change: 17 fold increase at 1 hr, p= 2.60E-13), and pleiotrophin, a mitogenic growth factor for fibroblast and endothelial cells (PTN; peak change: 4.7 fold increase at 4 hrs, p= 2.77E-10). Among proteins that increased after PMI, 21 were elevated in SMI as compared to non-ischemic controls at nominal significance (n=20, p < 0.05, Student’s t-test).
Conclusions: Our studies demonstrate a multiplexed nucleotide-labeled antibody-based proteomic assay can detect new biomarkers of myocardial injury. These findings motivate additional studies in large, heterogeneous patient cohorts with spontaneous coronary syndromes.