Aim: Cardiac macrophages represent a crucial cell population for maintaining heart function as they facilitate electrical conduction through the atrioventricular node and influence heart recovery post-MI through neuroimmune signaling. Therefore, characterization of macrophages in the heart based on their receptor profile is increasingly relevant, offering new clinical insights and enticing possibilities for the development of novel therapeutic strategies. 

 

Methods and results: Using a combined approach of single-cell, deep RNA sequencing, and immunofluorescence staining of cells isolated from mouse hearts, the β2-adrenoceptor was detected as highly and specifically expressed on macrophages. As the β2-specific agonist clenbuterol induces cardiomyocyte hypertrophy, we hypothesized that this effect might be mediated through activation of β2-adrenoceptors on cardiac macrophages. To investigate macrophage β2-adrenoceptor signaling and its impact on cardiomyocyte growth, we first assessed cAMP formation in macrophages transfected with a fluorescence resonance energy transfer-based cAMP sensor. Upon stimulation with β2-agonists, a substantial 2,7-fold change in intracellular cAMP concentration could be observed. Next, a co-culture system of primary isolated macrophages and adult (CM), as well as neonatal cardiomyocytes (NMCM), was established, and cardiomyocyte hypertrophy was determined by automated cell size measurement. The hypertrophy induction was four-times significantly higher in cardiomyocytes co-cultured with macrophages, with adult cardiomyocyte size of 9149,1 µm2 (±404,6 µm2) and neonatal cardiomyocyte size of 3992,2 µm2 (± 65,8 µm2), compared to adult cardiomyocyte monocultures (size: 6916,4 ± 252,6 µm2) or co-cultures with neonatal cardiomyocytes and β2-adrenoceptor-deficient macrophages (size: 2862,8 ± 31,7 µm2). To validate this effect in an in vivo model, we generated a mouse line with macrophage-specific deletion of the β2-adrenoceptor by crossbreeding Cx3cr1-Cre and Adrb2flox/flox mice. After verification of successful knockout of the β2-adrenoceptor in cardiac macrophages, clenbuterol was injected intraperitoneally into these mice and the Adrb2flox/flox mice as respective control. Quantification of cardiomyocyte dimensions by WGA staining of LV cryosections revealed a two-fold significant difference in cardiomyocyte cell size, 831,0 µm2 (± 30,7 µm2) in the control mice compared to 662,4 µm2 (± 1,9 µm2) in the Cx3cr1-Cre-Adrb2flox/flox group, suggesting that β2-adrenoceptor activation on macrophages indeed triggers cardiomyocyte growth.

 

Conclusion: In summary, we have identified the macrophage-specific expression of the β2-adrenoceptor in the heart and demonstrate a key role of the macrophage β2-adrenoceptor in cardiomyocyte hypertrophy. Additional studies investigating the effects of macrophage-specific β2-deficiency in cardiac disease models, including the application of beta-blockers, are warranted to evaluate the prospects of therapeutic intervention of macrophage β2-adrenoceptors.