Briefly, CD36-/-36 (link), LRPfl/fl LysMcre37 (link), and Mertk-/-38 (link) bone marrow-derived mouse macrophages were co-cultivated with primary adult mouse CMs and efferocytosis assessed ex vivo. Parallel assays were performed in which Mertk was reconstituted into cells to test sufficiency. In vivo, Mertk+/+ and Mertk-/- mice and bone marrow chimeras were subjected to myocardial infarction after permanent occlusion of the left anterior descending artery. Subsequently, Mertk expression (mRNA and protein), apoptotic cell accumulation (TUNEL), in situ efferocytosis, infarct size (TTC and collagen staining), inflammatory parameters (chemokine and cytokine mRNA and MΦ accumulation), and ventricular remodeling and cardiac function (echocardiography) were measured. The identification of glycosylated solMER in heart extracts was corroborated by addition of an N-glycanase. Statistical Analysis. Results are presented as means +/- SEM. Differences between multiple groups were compared by analysis of variance as appropriate and as indicated (2-way ANOVA and Bonferroni post-test), and differences between 2 groups were compared by unpaired Student t test (indicated by #). Two way repeated measures ANOVA was used to evaluate the statistical significance of data acquired from the same animal over multiple time points. A p value of < 0.05 was considered to be significant as indicated by * or #. Stated “n” values are biological replicates. Survival distributions were estimated using the Kaplan-Meier method and compared by the log-rank test.
An expanded and detailed Materials & Methods section is available insupplemental Online Data .
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