The effects of macrophages on cardiomyocyte calcium-handling function using in vitro culture models

Following myocardial infarction (MI), myocardial inflammation plays a crucial role in the pathogenesis of MI injury and macrophages are among the key cells activated during the initial phases of the host response regulating the healing process. While macrophages have emerged as attractive effectors in tissue injury and repair, the contribution of macrophages on cardiac cell function and survival is not fully understood due to complexity of the in vivo inflammatory microenvironment. Understanding the key cells involved and how they communicate with one another is of paramount importance for the development of effective clinical treatments. Here, novel in vitro myocardial inflammation models were developed to examine how both direct and indirect interactions with polarized macrophage subsets present in the post-MI microenvironment affect cardiomyocyte function. The indirect model using conditioned medium from polarized macrophage subsets allowed examination of the effects of macrophage-derived factors on stem cell-derived cardiomyocyte function for up to 3 days. The results from the indirect model demonstrated that pro-inflammatory macrophage-derived factors led to a significant downregulation of cardiac troponin T (cTnT) and sarcoplasmic/endoplasmic reticulum calcium ATPase (Serca2) gene expression. It also demonstrated that inhibition of macrophage-secreted matricellular protein, osteopontin (OPN), led to a significant decrease in cardiomyocyte store-operated calcium entry (SOCE). In the direct model, stem cell-derived cardiomyocytes were co-cultured with polarized macrophage subsets for up to 3 days. It was demonstrated that anti-inflammatory macrophages significantly increased cardiomyocyte Ca²⁺ fractional release while macrophages independent of their subtypes led to significant downregulation of SOCE response in cardiomyocytes. This study describes simplified and controlled in vitro myocardial inflammation models, which allow examination of potential beneficial and deleterious effects of macrophages on cardiomyocytes and vise versa. This can lead to our improved understanding of the inflammatory microenvironment post-MI, otherwise difficult to directly investigate in vivo or by using currently available in vitro models.