The nucleus is a central regulatory hub integrating biochemical and mechanical cues to coordinate gene expression and chromatin organization. In the heart, precise nuclear control supports contractile and metabolic balance across chambers exposed to distinct hemodynamic and energetic demands. Low-abundance transcription factors and chromatin-associated proteins are key to these processes, yet their characterization has been limited by the fibrous, mitochondria-rich composition of cardiac tissue and the narrow dynamic range of conventional proteomics.
To address these challenges, we applied a biochemical-centrifugation enrichment strategy coupled with high-resolution data-independent acquisition (DIA) mass spectrometry to define the chamber-resolved nuclear proteome of the mouse heart. This workflow enhances detection of low-abundance nuclear proteins while maintaining native compartmental context and quantitative precision. Nuclear fractions from left and right atria and ventricles (n = 4) were benchmarked against global proteomes, followed by integrated bioinformatic analyses and immunofluorescence microscopy validation of localization and abundance.
This approach identified > 6,300 proteins, including ~2,900 nuclear-enriched and ~1,300 nucleus-annotated (GO:0005634) proteins, providing extensive coverage of transcriptional regulators and structural complexes. A one-way limma-based ANOVA (FDR < 0.05) revealed 973 proteins with region-specific differences grouped into six dominant expression clusters. Specifically, oxidative- and TCA-linked regulators were enriched in the left ventricle, ECM–receptor and PI3K–Akt-associated proteins in the left atrium, and complement- and platelet-signaling proteins in the right atrium. Immunofluorescence confirmed these spatial patterns, with SUN2 and H2AC21 enriched in the left atrium and PTBP2 elevated in the left ventricle.
This study establishes the first chamber-resolved nuclear proteomic atlas of the mouse heart, capturing nuclear proteins within their active regulatory environment. It provides a spatially resolved framework for understanding nuclear specialization underlying regional cardiac function and identifies nuclear signaling pathways and transcriptional regulators that represent promising targets for future therapeutic intervention in heart disease.