Heart failure (HF) following reperfused myocardial infarction (repAMI) is caused by irreversible left ventricular (LV) cardiomyocyte loss as well as interstitial fibrosis during the remodeling process. During repAMI, mitochondria are severely compromised and contribute significantly to cell death through the release of ROS and activation of the NLRP3 inflammasome. In repAMI, structural and functional abnormalities in mitochondria disrupt their overall homeostasis and the NLRP3 inflammasome induces inflammatory processes. Both events drive disease progression by facilitating cardiomyocyte death. It is therefore necessary to preserve cellular homeostasis and cardiac function by efficiently removing damaged mitochondria. Dysfunctional mitochondria are removed via intracellular degradation pathways that involve autophagy and lysosomal degradation. These removal pathways are further supported by the extracellular clearance via macrophages. Cardiac resident macrophages with their phagocytic receptor MERTK remove damaged mitochondria from cardiomyocytes, however, all these clearance processes seem to be impaired during repAMI. This study investigates how impaired macrophage-cardiomyocyte signaling during repAMI contributes to the accumulation of dysfunctional mitochondria, autophagy blockage and ultimately, cardiomyocyte cell death.
In this study, we employed a repAMI mouse model. Transmission electron microscopy (TEM) was used to investigate the accumulation of damaged mitochondria over time. Immunofluorescence (IF) staining was performed to examine the activity of the NLRP3 inflammasome, autophagosomes (LC3) and cardiomyocyte stress (ANP) in the repAMI mouse model as well as human LVAD patient samples. Furthermore, the presence of cardiac resident (CCR2-) macrophages and recruited macrophages (CCR2+) in the repAMI heart was assessed by IF staining. Imaging was performed with confocal microscopy.
TEM analysis showed the accumulation of damaged mitochondria in repAMI mouse hearts. Immunofluorescence (IF) analysis demonstrated the development of a stressed cardiomyocyte subpopulation with NLRP3 inflammasome activation up to 28 d in repAMI. Stressed cardiomyocytes, localized to the border zone, exhibited NLRP3 inflammasome activation, whereas non-stressed areas showed no activation. CCR2- macrophages were absent in the border zone, while CCR2⁺ macrophages were predominantly observed in the infarct zone and persisted over time. The remote region displayed a macrophage distribution pattern similar to that of sham hearts used as control. Light-sheet microscopy revealed the three-dimensional spatial distribution of macrophages.
The activation of the NLRP3 inflammasome in stressed cardiomyocytes of repAMI mouse hearts is potentially driven by the accumulation of damaged mitochondria. CCR2- macrophages capable of clearing debris are absent in the border zone, further impairing clearing mechanisms of dysfunctional mitochondria and exacerbating cellular stress. Furthermore, the presence of CCR⁺ macrophages with MERTK receptor in the infarct zone suggests their involvement in clearing dead cells and promoting inflammation. These processes contribute to adverse cardiac remodeling, ultimately compromising cardiac function. The absence of macrophages around stressed cardiomyocytes in repAMI therefore represents a potential therapeutic target to mitigate the adverse cardiac remodeling and cardiac damage.
