Abstract

Pyroptosis, a caspase‐1‐dependent cell death mechanism, is upregulated in the diabetes mellitus (DM) heart. However, its underlying regulatory mechanisms are unclear. Here, we investigated the regulatory role of matrix metalloproteinase‐9 (MMP9), which is upregulated and promotes pathological remodeling, on pyroptosis of the DM heart. We hypothesized that upregulated MMP9 in the DM heart induces cardiac pyroptosis, which can be rescued by ablation of MMP9. To test the hypothesis, we created a novel DM mice strain where MMP9 gene is deleted (Ins2+/−/MMP9−/− or DKO), by crossbreeding Ins2+/− Akita (spontaneous, genetic T1DM model) male with MMP9−/− (MMP9KO) female mice. To assess the effect of DM on cardiac pyroptosis, we used Ins2+/− Akita and its sibling Ins2+/+ WT mice. For all experiments, we used fourteen‐week male mice. For in vitro studies, we used HL1 cells (cardiomyocyte cell line of murine origin) and generated MMP9−/− HL1 cells by CRISPER/Cas9 method. We treated HL1 and MMP9−/− HL1 cells with normal (NG= 5 mM) or high (HG= 25 mM) doses of D‐glucose for 24 hours. We measured the activity of MMP9 by in‐situ and in‐gel gelatin zymography. For pyroptosis, we measured the protein levels of NLRP3 (involved in the inflammasome complex formation that is required for pyroptosis initiation), caspase‐1 (the key enzyme for pyroptosis), and interleukine‐1β (IL‐1β) and interleukine‐18 (IL‐18) (the pro‐inflammatory cytokines that are cleaved by caspase‐1) by Western blotting. We corroborated cell death by pyroptosis using LDH release assay in vitro. We found absence of MMP9 activity in DKO hearts and MMP9−/− HL1 cardiomyocytes, and upregulation of MMP9 in Akita hearts and hyperglycemic HL1 cardiomyocytes, which supports our published reports and validate our model system. The densitometric analyses of Western blot bands revealed that pyroptosis markers are upregulated in Akita hearts but they are decreased in DKO hearts. The protein levels (mean± SE) of different pyroptosis markers in the heart are: NLRP3: WT 1.6 ± 0.2, Akita 2.5 ± 0.2, DKO 0.4 ± 0.0; cleaved/total caspase‐1: WT 0.2 ± 0.0, Akita 0.3 ± 0.0, DKO 0.2 ± 0.0; cleaved/total IL‐1β: WT 0.3 ± 0.0, Akita 0.5 ± 0.0, DKO 0.1 ± 0.0; cleaved IL‐18: WT 0.4 ± 0.0, Akita 1.0 ± 0.0, DKO 0.2 ± 0.0 (decimal values are rounded). The in vitro studies showed similar patterns of protein levels (mean± SE): NLRP3: HL1+NG 0.08 ± 0.02, HL1+HG 0.14 ± 0.01, MMP9−/− HL1+HG 0.08 ± 0.00; caspase‐1: HL1+NG 0.6 ± 0.1, HL1+HG 1.0 ± 0.08, MMP9−/− HL1+HG 0.40 ± 0.05; IL‐1β: HL‐1+NG 0.60 ± 0.03, HL‐1+HG 0.80 ± 0.04, MMP9−/− HL1+HG 0.10 ± 0.01; IL‐18: HL‐1+NG 1.60 ± 0.20, HL‐1+HG 2.40± 0.20, MMP9−/− HL1+HG 0.20 ± 0.03. The LDH release assay showed that high‐glucose treatment causes increased death of cardiomyocytes (absorbance at 490–690 nm for red formazan formation: HL1+NG 0.20 ± 0.01, HL1+HG 0.40 ± 0.00), which was decreased by ablation of MMP9 (MMP9−/− HL1+HG 0.10 ± 0.01). In conclusion, we have demonstrated for the first time that MMP9 inhibition could alleviate pyroptosis of the DM heart.