GM6001 and MMP9/MMP13 inhibitor I treatment, and chemokine injection

GM6001 (ab120845; Abcam) and MMP9/MMP13 inhibitor I (21265; Cayman) were prepared at a stock concentration of 20 mM in DMSO. Just prior to use, the GM6001 or MMP9/MMP13 inhibitor I stock solution was diluted to 100 μM with Hanks’ solution. Zebrafish were anesthetized and then injected intraperitoneally with 10 μL of the 100 μM GM6001 or MMP9/MMP13 inhibitor I solution. A control group was injected with 10 μL Hanks’ solution containing 0.5% DMSO alone. In one series of experiments, zebrafish were injected with GM6001 (or DMSO) once a day for up to 30 days. In another series of experiments, zebrafish were split into three groups: one group was injected with DMSO alone for 14 days; another group was injected with GM6001 for 7 days and DMSO for the following 7 days; and the final group was injected with DMSO for 7 days and then with GM6001 for the next 7 days. Immediately after the first injection, the heart was cryoinjured, as described above.

In one series of experiments, following cryoinjury and GM6001 treatment, the densities of leukocytes and the expression of the cxcl8 and ccl2 genes were measured at various time points. Intact hearts were harvested from fish treated for 24 hours with DMSO or GM6001 and used as the control group.

In another series of experiments, following cryoinjury and GM6001 treatment, anesthetized zebrafish were microinjected into the injured area of the ventricle with a ~15 nL mixture of CXCL8 and CCL2 (at 250 μg/mL and 650 μg/mL in PBS, respectively). Zebrafish were injected with these chemokines twice a day from 1 dpc to 4 dpc, and then once a day from 5 dpc to 7dpc. Control fish were cryoinjured and treated with GM6001, but injected with PBS only. In an additional series of control experiments, cryoinjured fish without GM6001 treatment were injected with CXCL8 and CCL2 individually or in combination into the injured area. The hearts were harvested at different time points, and the volume of the scar as well as the numbers of neutrophils and macrophages were quantified.