Supplementary Figure 6: Resident cardiac macrophages proliferate post myocardial infarction in the remote myocardium, with resident macrophage depletion leading to increased cardiomyocyte hypertrophy without cardiac fibrosis.

3-week-old Cx3cr1^CreER–YFP:R26^Td mice were fed tamoxifen (TAM)- containing chow for 10 days, and then mice were either left as non-infarcted controls for an additional 6 weeks. a) Diphtheria toxin (DT) was then administered to Cx3cr1^CreER–YFP:R26^Td/DTR mice for 2 weeks at steady state. Blood monocytes (CD115⁺CD11b⁺Ly6C⁺), neutrophils (CD45⁺CD11b⁺CD64^–Ly6G⁺), lymphocytes (CD45⁺CD11b⁺CD64^–), Td^–CCR2⁺ cardiac macrophages, and Td⁺ cardiac macrophages were quantified by flow cytometry, normalized to DT-injected Cx3cr1^+/+:R26^Td/DTR control mice. N=5; repeated independently 3 times with similar results. b) Representative flow plots of resident macrophage depletion during steady state (Fig. 6a) from blood and cardiac tissue following a myocardial infarction demonstrating effective depletion of resident macrophages. c) Naive wild-type mice were given chronic PBS or 250ng DT injections (i.p.) for 35 days. Whole hearts were either cut longitudinally and sectioned to be stained with Fast Green for interstitial fibrosis, quantified as % Fibrosis using ImageJ software, or quantified by flow cytometry for the number of neutrophils or cardiac macrophages normalized per mg tissue. N=3, 7; one experiment. d) Hearts were isolated from adult TAM-fed Cx3cr1^CreER–YFP:R26^Td/DTR mice (as above) day 5 post-MI, comparing non-depleted or depleted mice (DT treated daily). Recruited macrophages (MF), monocytes, and neutrophils were quantified and normalized per mg tissue in the ischemic and remote cardiac zones. N=6,5,4; repeated independently 2 times with similar results. e-f) Hearts were isolated from adult Cx3cr1^CreER–YFP:R26^Td mice following TAM-induction (as above), and mice received BrdU injection 2 hours prior to harvest. Resident MFs (Td⁺), recruited MFs (Td^–) and monocytes were normalized per mg tissue in the remote zone at various time points (e), or %BrdU⁺ was determined (f), compared to an uninfarcted control (No MI). N(Td⁺)=13, 13, 6, 3, 10. N(Td^–, mono)= 12, 10, 7, 3, 6; Repeated 3 times for day 2, 2 times for day 4 and 28, 1 time for day 7, 6 times for control with similar results. g) Change in fractional shortening (contractile function) from day 7 to day 28 post-MI in the remote zone comparing mice with or without chronic resident macrophage depletion (Paired T-test) through the use of M-mode echocardiography. N=11; repeated 2 times independently with similar results. h-i) Longitudinally cut hearts from Cx3cr1^CreER–YFP:R26^Td/DTR mice (as above) isolated day 35 post-MI to compare post-MI control and depleted groups. Cardiomyocyte hypertrophy was detected using Wheat Germ Agglutinin staining in the remote zone. Hearts imaged at 20x magnification and cardiomyocytes measured in ImageJ, 8 images/zone/heart at 2 cutting levels. N=4, 6, 6; repeated once with similar results (h); Cardiac tissue fibrosis was detected using Fast Green staining. Percentage fibrotic area was quantified with ImageJ analysis, 4 images/zone/heart at 2 cutting levels. N = 9; repeated once with similar results. For all: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; Two-tailed Student’s T test; center value, mean; error bars, SEM.