Cardiac macrophages support myocardial function in human heart failure

Background: Cardiac macrophages play distinct roles in both heart health and disease, positioning them as potential targets for immunotherapy. However, their precise functions in human heart failure (HF) remain unclear due to the inability to test macrophage interventions in living human tissue.

Purpose: This study investigated the role of human macrophages in long-term cultured, living heart tissue from patients with HF.

Methods: Explanted hearts from 11 patients with HF were used to obtain human left ventricular myocardial tissue slices for long-term in vitro culture. Cardiac macrophages were depleted by treating the slices with Csf1 receptor inhibitor PLX-5622 (Csf1Ri) for 10 days. Contractile function was continuously monitored throughout the treatment period. Following treatment, tissue slices were paraffin-embedded and stained for CD68 to assess the effectiveness of macrophage depletion. Caspase-3 assay and TUNEL staining were performed to evaluate cardiomyocyte apoptosis. Mitochondrial function was evaluated via Complex IV and AmplexRed assays, transmission electron microscopy (TEM), and high-resolution respirometry (Oroboros-O2k).

Results: CD68 staining confirmed a significant reduction in macrophages in Csf1Ri-treated human myocardial tissue, indicating effective depletion (n=5 slices each, p=0.0317). Continuously paced, macrophage-depleted human slices (n=35) showed a progressive decline in systolic function, with significantly lower force amplitudes compared to controls (n=33). Functional contractility assessments via force-frequency relationship and post-rest potentiation suggested disrupted sarcoplasmic reticulum calcium handling and resulting cardiomyocyte dysfunction following macrophage loss. Macrophage-depleted heart tissue showed increased cardiomyocyte apoptosis, as demonstrated by elevated Caspase-3 activity (n=10 slices each, p=0.0304) and enhanced TUNEL staining (n=5 slices each, p=0.0047), suggesting cardiomyocyte apoptosis as potential contributor to contractile dysfunction. We therefore performed further structural analyses. TEM imaging revealed significant mitochondrial abnormalities, including increased mitochondrial size (n=55 FOV each, p<0.0001), higher proportion of structurally abnormal mitochondria (n=400 FOV each, p<0.0001), reduced cristae density (n=50 FOV each, p<0.0001) and impaired electron density (n=50 FOV each, p<0.0001) in macrophage-depleted tissue. Further functional analysis by high-resolution respirometry showed mitochondrial dysfunction in macrophage-depleted slices. We detected a significantly reduced complex I related (n=9 slices each, p=0.0420) and maximum oxidative phosphorylation capacity (n=8 each, p=0.007). Reduced maximum activity of the respiratory system was still detectable after uncoupling, clearly indicating dysfunction of the whole respiratory system. (ETS, n=8 each, p=0.0037). As a consequence, we found increased levels of oxidative stress following macrophage depletion, as measured in culture supernatants on day 10 of treatment (n=20 slices each, p=0.0371).

Conclusions: Macrophage depletion in tissue from patients with HF causes a decline in systolic force, likely driven by mitochondrial dysfunction and cardiomyocyte apoptosis. These findings provide the first functional evidence in human heart tissue that macrophages are essential for maintaining myocardial tissue homeostasis and contractility in HF, which has important implications for potential cardiac immunotherapy.