Unbiased complexome profiling and global proteomics analysis reveals mitochondrial impairment and potential changes at the intercalated disk in presymptomatic R14Δ/+ mice hearts

Background Phospholamban (PLN) is a sarco-endoplasmic reticulum (SER) membrane protein that regulates cardiac contraction/relaxation by reversibly inhibiting the SERCA2a Ca2+-reuptake pump. The R14Δ-PLN mutation causes severe cardiomyopathy that is resistant to conventional treatment. Protein complexes and higher-order supercomplexes such as intercalated disk components and Ca+[2][1]-cycling domains underlie many critical cardiac functions, a subset of which may be disrupted by R14Δ-PLN. Methods We developed an improved complexome profiling (CP) workflow specifically geared towards identifying disruption of very high molecular-weight (>2 MDa) protein complexes and supercomplexes in presymptomatic R14Δ/+ mice hearts. Ventricular tissues were homogenized under non-denaturing conditions, fractionated by size-exclusion chromatography (SEC) and subjected to quantitative data-independent acquisition mass spectrometry (DIA-MS) proteomics analysis. Systematic analysis of CP data using conventional strategies yielded limited insights, likely due to underrepresentation of cardiac-specific complexes in the curated protein complex databases used as ground-truth for analysis. We thus developed PERCOM: a novel data analysis strategy that does not rely upon protein complex databases and can, furthermore, be implemented on widely available spreadsheet software. Results SEC-DIA-MS coupled with PERCOM identified 296 proteins with disrupted elution profiles in presymptomatic 28wk-old R14Δ/+ mice. Hits were significantly enriched for mitochondrial and intercalated disk (ICD) components. Alterations to mitochondrial and ICD supercomplexes were observed in mice as young as 9wks of age and were associated with reduced expression of mitochondrial proteins and maximal oxygen consumption rate. Conclusion Using a novel CP workflow, we identify mitochondrial alterations as an early-stage R14Δ-PLN event and provide preliminary data showing effects at the ICD. These molecular components underlie critical cardiac functions and their alteration at a young age may contribute to R14Δ-PLN pathogenesis. ### Competing Interest Statement The institution of Drs. De Boer and Sillje has received research grants and/or fees from AstraZeneca, Abbott, Boehringer Ingelheim, Cardior Pharmaceuticals GmbH, Ionis Pharmaceuticals. Novo Nordisk, and Roche; Dr. de Boer has had speaker engagements with and/or received fees from and/or served on an advisory board for Abbott, AstraZeneca, Bristol Myers Squibb, Cardior Pharmaceuticals GmbH, NovoNordisk, and Roche; Dr. de Boer received travel support from Abbott, Cardior Pharmaceuticals GmbH, and NovoNordisk. [1]: #ref-2