Unbiased complexome profiling reveals altered mitochondrial and intercalated disk supercomplexes in presymptomatic R14 2 Δ/+ mice hearts

ABSTRACT: Background: Phospholamban (PLN) is a tail-anchored sarco-endoplasmic reticulum (SER) membrane protein that regulates cardiac contraction/relaxation by reversibly inhibiting the SERCA2a Ca2+ 36 -reuptake pump. The R14Δ-PLN mutation causes severe cardiomyopathy that is resistant to conventional treatment. Multi-protein complexes and higher-order supercomplexes, such as intercalated disk components and SER Ca+2 -cycling domains, underlie many critical cardiac functions, a subset of which may be disrupted by R14∆-PLN. Complexome profiling (CP) is a systems biology approach to characterize high molecular weight (MW) assemblies in biological samples. Most current CP data analysis strategies rely on external protein42 complex databases that are predominantly curated from immortalized or cancerous cell lines; thus, assemblies unique to specialized cell types such as cardiomyocytes, as well as higher-order supercomplexes that may have fluid stoichiometry/composition are significantly underrepresented. The requirement for computer programming expertise also presents a barrier to generalized adoption. Methods: We developed and applied a novel CP workflow to identify protein-complexes/supercomplexes disrupted in presymptomatic 28wk-old R14Δ/+ knockin mice hearts. Cardiac tissues were homogenized under non-denaturing conditions, fractionated by size-exclusion chromatography (SEC) and subjected to quantitative data-independent acquisition mass spectrometry (DIA-MS)-based proteomics analysis. As expected, traditional database-dependent analysis offered only limited insights due to underrepresentation of cardiac-specific assemblies and higher-order supercomplexes. We thus developed a novel data analysis strategy (PERCOM) that does not rely upon external protein-complex databases and can, furthermore, be implemented on widely available spreadsheet software. Results: SEC-DIA-MS coupled with PERCOM analysis identified 296 proteins with disrupted MW-distribution profiles. Hits were significantly enriched for mitochondrial and intercalated disk (ICD) components. Mitochondrial alterations were confirmed at the level of global protein expression and metabolic function. Disruption of mitochondrial and ICD supercomplexes was confirmed in mice as young as 9wks of age. Conclusion: We present a novel CP workflow that is highly suited to the analysis of very high MW protein-complexes and supercomplexes in specialized tissue types. It includes a data analysis strategy that removes a computer programming resource barrier to general application. Our findings are among the first to show altered mitochondrial function as a hallmark of early-stage R14Δ-PLN, and identify disruption of the intercalated disk as a novel potential disease pathway. These molecular-level alterations target key machinery underlying cardiac energy generation and electromechanical connectivity, and may significantly contribute to the R14∆-PLN pathogenesis.

INSTRUMENT(S): timsTOF Pro 2

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Heart

DISEASE(S): Cardiomyopathy

SUBMITTER: Christof Lenz  

LAB HEAD: Christof Lenz

PROVIDER: PXD050383 | Pride | 2024-09-26

REPOSITORIES: Pride