(P126) MASS SPETROMETRY EVIDENCE FOR THE MULTI-SITE PHOSPHORYLATION OF CARDIAC RYANODINE RECEPTOR 2 IN ATRIAL FIBRILLATION

Background: Atrial fibrillation (AF) is the most common sustained arrhythmia. Currently, there are no biomarkers available for monitoring AF progression or for serving as targets for disease-modifying therapies. Research in this area is increasingly pointing to a role for post-translational phosphorylation of cardiac ryanodine receptor 2 (RyR2) as a promising avenue for biomarker discovery. Most studies of RyR2 phosphorylation in AF have focused on single phosphorylation sites using antibody-based assays. Here, we use mass spectrometry (MS) to perform a comprehensive assessment of multiple RyR2 phosphorylation sites simultaneously in human right atrial samples collected from patients with AF and healthy controls.

METHODS AND RESULTS: Consenting participants in the Cardiac Biobank Registry of St. Paul’s Hospital in British Columbia were screened for eligibility. This registry contains cardiac tissue samples collected during open heart surgery. 351 patients were included for screening. The criteria used for AF patient samples was documented persistent or permanent AF on chart review. Control samples consisted of patients with no AF on chart review. All AF and control patients were subsequently screened for the following criteria: (1) sample collection confirmed to be from right atrial tissue, (2) at least 40mg of sample available, and (3) a sample ischemic time of ≤5mins. AF and control patients were matched for age, sex, left ventricular ejection fraction (LVEF), and cardiac risk factors. Based on these criteria, 7 AF and matched controls were eligible for MS analysis. Patient demographics are included in Table 1. Right atrial tissue from AF and control patients were dissociated and enzymatically prepared for MS analysis. Next, we performed MS on prepared samples with a focus on RyR2 phosphorylation sites. Data is currently available from 5 AF and matched control patients and a summary of key phosphopeptides is reported in Figure 1. We find evidence of multi-site phosphorylation of RyR2 in persistent and permanent AF, notably at S2808, S2811, and S2814. Previous data from our group has shown that these residues can be phosphorylated by both protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII), with the latter being more efficient at phosphorylating multiple sites.

Conclusion: We provide preliminary evidence for the multi-site phosphorylation of RyR2 in persistent and permanent AF. This suggests that the post-translational modification of RyR2 in longstanding AF is dynamic and potentially graded. Although CaMKII is the most likely candidate, establishing the order of phosphorylation and the exact kinases involved can be an area for future research.