Chamber-resolved nuclear proteome atlas of the ischemic heart

Background Early events following myocardial ischemia–reperfusion (I/R) represent a critical therapeutic window. Nuclear reprogramming is central to cellular phenotype and tissue homeostasis, yet remodelling of the cardiac nuclei during this phase is poorly understood. Purpose To generate a chamber-resolved cardiac nuclear proteome for the characterisation of early remodelling after I/R. This will enable resolution of molecular targets at a subcellular and regional level for cardiac repair. Methods We optimised a chamber-specific nuclear-enrichment proteomics workflow for the heart. In the I/R model, C57BL/6 mice underwent transient coronary ligation followed by 4 h reperfusion or sham surgery. Cardiac tissues were subjected to subcellular fractionation followed by high-sensitivity mass spectrometry-based proteomics. Nuclear enrichment and regional localisation of selected proteins were validated on heart by immunofluorescence microscopy. Results We defined a novel nuclear proteome signature of the heart and its regions. In sham hearts, we resolved 2,876 nuclear-enriched protein groups across cardiac chambers, defining region-resolved nuclear signatures enriched for RNA processing, chromatin regulation, nuclear envelope/lamina, proteostasis, stress-response and cell-death-related programs. Importantly, male left ventricle (LV) nuclei, early (4 h) I/R induced marked proteome remodelling, with >250 proteins increased and ~30 decreased versus sham with limited remodelling in corresponding female group. Differentially expressed proteins included stress-responsive transcriptional regulators, DNA-damage and chromatin-associated factors, RNA-regulatory proteins, and nucleo–cytoskeletal components with reported nuclear or perinuclear roles, consistent with rapid activation of nuclear stress-response and remodelling pathways in the reperfused LV. Conclusion This optimised nuclear-enrichment and proteomic workflow enabled a high-coverage, chamber-resolved mapping of the cardiac nuclear proteome and revealed selective early remodelling of the LV nuclear landscape after IR. These insights provide a framework for linking nuclear proteomic changes to injury and will prioritise candidate pathways for cardioprotective intervention within the acute reperfusion window.