Abstract Body (Do not enter title and authors here): Background: While chronic pathological substrates of sudden cardiac death (SCD) are well-known, the molecular triggers that predispose individuals to fatal arrhythmias that day remain undefined, posing a barrier to near-term prevention. We recently reported the first transcriptome study of human myocardium sampled at autopsy-confirmed SCD to demonstrate upregulation of active fibrosis and selected channel dysregulation as a vulnerable substrate for lethal arrhythmias via candidate gene approach. Here, the study of comprehensive proteomes in autopsy-confirmed SCD samples enables unbiased discovery of novel pro-arrhythmic pathways and biomarkers.

Hypothesis: The paired myocardium and circulating blood of autopsy-defined SCDs exhibit distinct proteomes compared to non-cardiac sudden deaths and trauma deaths that reflect their vulnerable myocardial state and its associated circulating biomarkers in the hours to days before SCD.

Aims: Define the proteome of coupled myocardial tissue, neat serum, and enriched serum of autopsy-confirmed sudden deaths.

Methods: We conducted unbiased, quantitative proteomics via LC-MS on paired LV myocardium and serum samples from 13 arrhythmic SCDs (5 acute MI, 3 CAD, 3 DCM, 2 LVH) vs. 7 non-arrhythmic sudden deaths (occult overdose, myocardial rupture, and traumas) to define the arrhythmic substrate.

Results: Comprehensive proteomics resolved 5,979 proteins from myocardial tissue and 6,013 proteins from serum, representing a 3-fold increase in proteome depth vs. previous reports. Among these ~6,000 proteins, 2,088 were upregulated and 662 downregulated in Arrhythmic vs. Non-arrhythmic deaths. Arrhythmic myocardium exhibited 3-fold higher fibrosis-associated proteins (POSTN, LUM, FMOD, COL1A1) and 2-fold lower contraction-related proteins (TNNC1, TNNI3, MYLK3, MYL4). Our novel enrichment method resolved 3,645 proteins otherwise undetectable in the neat fraction (POSTN and COL1A1). Reactome analysis of overlapping differentially expressed proteins in myocardium and enriched serum revealed coordinated upregulation of extracellular matrix remodeling, metabolic dysfunction, and immune response.

Conclusion: The proteome of myocardium and serum at SCD reveals systemic and cardiac-specific molecular changes which together may exacerbate existing fibrosis and contribute to electrical instability and acutely vulnerable arrhythmogenic substrate. These findings provide candidate biomarkers for near-term risk assessment of fatal arrhythmias.