Effect of donor MI on BMC therapeutic efficacy. Donor BMCs were harvested from healthy and 3 day post-MI mice. Recipient and donor mice randomly underwent a parasternotomy MI by the left anterior descending artery ligation ~3 mm below the tip of the left atrium. All data are mean ± SD. (A) Recipient EF. Healthy donor BMCs significantly improved recipient 28 day post-MI EF. However, 28 day post-MI EF in the infarcted donor group was only preserved and significantly different from the healthy donor group. Recipient 28 day post-MI EF in the negative control HBSS group was significantly reduced and significantly different from the healthy and infarcted donor groups. Data are summarized numerically in Table S1. (B) Recipient ESV. (C) Recipient EDV. (D) Recipient anterior wall thickness at border zone and infarct area. (E) Recipient infarct size.

Implanted donor BMCs in recipient infarcted hearts. BMCs harvested from GFP⁺ transgenic mice under healthy and 3 day post-MI conditions were injected into myocardium of 3 day post-MI wild-type mice. (A) By detection of endogenous GFP fluorescence and immunofluorescence staining for GFP using a far-red secondary antibody and no red fluorophores, GFP⁺ cells were identified as those visible only by green and far red fluorescence but not red fluorescence. (B) Confocal imaging reveals two categories of GFP⁺ BMC morphology, round and irregular. (C) Ki-67 immunofluorescence (magenta) revealed proliferation in a subset of round and irregular GFP⁺ BMCs. Arrows show proliferating GFP⁺ BMCs. (D) Percentage distribution of proliferating and total (including non-proliferating) round and irregular GFP⁺ BMCs of healthy and infarcted donors on days 3 and 14 post-injection. (E) Examples of CD45⁺ and CD45^- implanted BMCs, taken from (left to right) MI d3, healthy d14, and two examples from MI d14. For these images only, brightness was optimized to demonstrate presence or absence of stain, rather than keeping it constant for all images. Green=GFP; red=CD45.

Influence of donor MI severity on BMC therapeutic impairment. All data are mean ± SD. (A) Injection of BMCs from small-MI donors led to preservation of recipient EF on day 28 post-MI; whereas injection of BMCs from large-MI donors resulted in a significant decrease in the 28 day post-MI EF and there was a significant difference between small and large MI donor groups (P<0.05). Moreover, the injection of BMCs from healthy donors led to recipient 28 day post-MI EF that was significantly higher than that of the small and large MI donor BMC groups and the HBSS group. The recipient 28 day post-MI EF in the small and large MI donor BMC groups was significantly higher than that of the HBSS group (P<0.05). (B) Thoracotomy MI led to the same reduction in BMC therapeutic efficacy as the original parasternotomy MI and the sham surgery slightly reduced the BMC therapeutic efficacy. The data are summarized numerically in Table S2.

MI-induced systemic inflammatory changes and alteration in bone marrow composition. (A) BMCs from healthy, sham, or post-MI donors (3 days post-MI/sham) were analyzed by flow cytometry. Data is representative of two independent experiments, each involving 3-4 mice/condition. For each parameter, histograms contain a representative curve from individual healthy (red), sham (blue), or post-MI (green) donors in the live gate. Each graph represents the results of 6-7 mice/condition. Error bars = SEM. (B) Contour plot depicts frequency of CD19⁺ B cells for individual mice. The right-most histogram depicts MHC-II expression on CD19⁺ cells from the contour plot. Graphs are as in A. (C) Representative histograms of CD11b, Gr1 and Ly6C^high of BMCs in the live gate. Tracings from two post-MI mice are shown due to increased heterogeneity in this group. Graphs are as in A. (D) MHC-II expression on myeloid cells. (E) Cytospin preparations of BMCs of healthy and 3 day post-MI donor mice. Results are representative of three samples/group. (F) Representative spleen histology. Increased erythropoiesis is evident as dense collections of darkly staining nuclei in red pulp at upper left in post-MI image and higher-power view shows increased erythroid cells and scattered granulocytes post-MI.

Correlations between BMC impairment and inflammatory response post-donor-MI. All data are mean ± SD. (A) Time courses of BMC impairment post-donor-MI. Therapeutic changes in BMCs were increasingly impaired over the first 3 days post-donor-MI but were less impaired after 7 days. * P<0.05 (no MI donors versus 1d, 3d, 5d, 7d and 21d post-MI donors; 1h post-MI donors versus 3d and 5d post-MI donors); # P<0.05 (no MI donors versus 3d, 5d and 21d post-MI donors). (B) Development of localized myocardial inflammation post-donor-MI detected by hematoxylin and eosin staining. Note that representative sections shown are from border zone rather than infarct scar. (C) Increased changes in pro-inflammatory cytokines and other proteins at varying times post-donor-MI in serum. Abbreviations of cytokines are shown in fig. S2. (D) A summarized illustration originated from (A), (B) and (C) shows a consistent correlation between time courses post-donor-MI of BMC therapeutic impairment and subsequent restoration (red line, EF), appearance of donor localized myocardial inflammation (black line, percent volume of LV), and elevation and subsequent reduction in serum levels of increased pro-inflammatory cytokines and other proteins (thinner black lines). Because these are relative, units are not shown on the Y-axis.

Prevention of BMC impairment by injection of anti-inflammatory drug or inhibitor to MI donor mice. All data are mean ± SD. (A) Preventive effect of dexamethasone administrated to post-MI donor mice on BMC impairment. BMCs were harvested from 3 day post-donor-MI mice that were administered dexamethasone or DMSO (vehicle) immediately after the left anterior descending artery ligation, and 24 and 48 hours post-MI. (B) Preventive effect of IL-1Ra injected into donor mice pre- and post-MI on BMC impairment. BMCs were harvested from 3 day post-donor-MI mice that received IL-1Ra or H₂O (vehicle) daily for 5 days (2 days pre-MI, 1 hour pre-MI, and 24 and 48 hours post-MI).