Unbiased complexome profiling and global proteomics analysis reveals mitochondrial impairment and potential changes at the intercalated disk in presymptomatic R14Delta/+ 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 R14Delta-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-cycling domains underlie many critical cardiac functions, a subset of which may be disrupted by R14Delta-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 R14Delta/+ 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 R14Delta/+ 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 R14Delta-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 R14Delta-PLN pathogenesis.