Background:
Acute myocardial infarction (AMI) often leads to long-term heart failure (HF) due to irreversible loss of left ventricular (LV) cardiomyocytes and interstitial fibrosis during cardiac remodeling. Preserving cardiomyocyte function and limiting adverse fibrotic remodeling are therefore critical to prevent LV dysfunction and HF progression. Recent single-cell transcriptomic studies have revealed distinct subpopulations of LV cardiomyocytes that emerge after myocardial infarction, characterized by cellular stress resulting from reperfusion, metabolic, and microenvironmental alterations following reperfused AMI (repAMI). While these subpopulations appear to differentially influence post-infarction healing and ventricular remodeling, their role remains poorly understood. A characterization of these subpopulations is essential to understand the mechanisms driving fibrosis formation and adverse cardiac remodeling after repAMI.
Methods:
We identified and characterized stressed cardiomyocyte subpopulation at the protein level in myocardial samples from end-stage HF patients receiving left ventrical assistant device (LVAD) implantation and in a mouse repAMI model. Immunofluorescence confocal microscopy was used to localize this subpopulation within the ventricle, while 3D light sheet microscopy enabled spatial distribution analysis. Fibrosis was visualized using Picrosirius red staining at multiple time points in repAMI, and their spatial relationship to stressed cardiomyocyte subpopulation was determined by colocalisation with immunofluorescence. We assessed the temporal and spatial dynamics of stressed cardiomyocytes throughout 28-days in repAMI. Additionally, we performed laser microdissection (LMD) coupled with mass spectrometry-based proteomics to isolate stressed cardiomyocytes from the mouse LV with spatial precision and identify their unique protein expression profiles. This proteomic signature enables a direct comparison with human patient samples.
Results:
Stressed cardiomyocytes were consistently localized in the border zone of the infarct. Although their abundance fluctuated over time, these cells remained persistently present in the border zone throughout the healing process, detectable up to 28 days in reperfusion. Picrosirius red staining revealed close spatial association between the stressed cardiomyocyte subpopulation and fibrotic regions. LMD-based proteomic analysis identified distinct molecular signatures specific to these stressed cardiomyocytes. These findings suggest that stressed cardiomyocytes actively influence cardiac remodeling and fibrosis progression during cardiac remodeling.
Conclusions:
This study demonstrates the persistent presence of the stressed cardiomyocyte subpopulation in the border zone and their close spatial association with fibrosis during cardiac remodeling following repAMI. Through spatial imaging and LMD proteomics, we revealed cell-type-specific molecular alterations indicating that stressed cardiomyocytes function as active mediators in post-infarction.
