Unbiased complexome profiling and global proteomics analysis reveals mitochondrial impairment and potential changes at the intercalated disk in presymptomatic R14^Δ/+mice hearts

Abstract

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.

mini-Complexome Profiling (mCP), an FDR-controlled workflow for global targeted detection of protein complexes

“…the analysis of cardiomyocytes obtained from different cavities of an individual adult wild-type mouse heart in the C57BL6N genetic background. Previous work from both our lab (Foo et al, 2024) and other groups (Rosca et al, 2008; Gómez et al, 2009; Hou et al, 2019) has shown PCC and higher-order protein supercomplexes to play key roles in the heart, ranging from energy production (i.e. mitochondrial CI/III/IV supercomplexes) to Ca +2 -handling (Alsina et al 2019).…”

“…as employed by Schulte et al (Schulte et al, 2023)) combined with high fraction numbers, and (ii) publicly available PPC databases are largely derived from the analysis of human cancer research cell lines, while organ-specific proteomes from other species such as the mouse heart are strongly underrepresented e.g. CORUM 4.0 contains <1% of entries curated from tissue or cardiomyocyte samples (Foo et al, 2024).…”

“…3C). We have begun to leverage this advantage for the analysis of very high MW, membrane-associated functional protein complexes that may exist as dynamic, non-covalent ‘functional rafts’ rather than as defined stoichiometric complexes (Foo et al, 2024). While it may be impossible to call out defined complex stoichiometries under these circumstances, differential analysis may nonetheless allow us to detect biochemical changes in large-scale complexome comparisons.…”

“…Alternatively, enriched membrane fractions may be solubilized by glycosides such as digitonin, a strategy frequently used for the detection of mitochondrial (Schtigger et al, 1991) or cardiac membrane proteins (Alsina et al, 2019). E.g., mitochondrial supercomplexes were visualized using blue native electrophoresis (BNE) following mild digitonin or Nonoxinol 40 (NP-40) extraction (Acín-Pérez et al, 2008), or using size exclusion chromatography (SEC) followed by octaethylene glycol monododecyl ether (C12E8) extraction (Foo et al, 2024). In the context of cardiac research, the compositions of active mitochondrial human respiratory super-complexes composed of mitochondrial complex I, III, and IV (CI1III2IV1) and the architecture of mitochondrial complex I, II, and IV(CI2III2IV2) were elucidated (Guo et al, 2017).…”