Abstract

Autophagy is a lysosome‐mediated catabolic process for recycling of defective cytoplasmic components. Matrix metalloproteinase‐9 (MMP9) is a collagenase, and ablation of MMP9 is cardioprotective for diabetic hearts. However, whether the cardioprotective role of MMP9 ablation is mediated by autophagic adaptation is unclear. We hypothesized that ablation of MMP9 protects the diabetic heart by upregulating cardiac autophagic flux. To test the hypothesis, we used 12–14 week male WT (C57 BL/6J), Ins2+⁄− Akita (Type1 diabetic), MMP9 knockout (MMP9−⁄−), and Ins2+⁄−/MMP9−⁄− (DKO) mice. DKO mice were generated by cross‐breeding male Ins2+⁄− Akita with female MMP9−⁄− mice. We measured the levels of LC3B‐II protein accumulation (autophagic flux marker) in these mice using bafilomycin A1 (inhibitor of autophagic degradation). To evaluate the cardiac function, we performed M‐mode echocardiography. For in vitro studies, we generated stable MMP9−⁄− HL1 cardiomyocyte cell line using CRISPR‐cas9 technique (MMP9−⁄− HL1). Autophagic flux was measured in control and MMP9−⁄− HL1 cardiomyocytes treated with low (LG‐5mM) or high (HG‐25mM) dose of D‐glucose. Our results revealed that autophagic flux is upregulated in MMP9−⁄− hearts (1.44±0.07 fold) as compared to WT hearts. Autophagic flux was also upregulated in Ins2+⁄− Akita hearts (1.3±0.12 fold) as compared to WT hearts. However, ablation of MMP9 in Ins2+⁄− Akita further increases cardiac autophagic flux (1.75±0.25 fold of Akita). The in vitro studies demonstrated that ablation of MMP9 in HL1 cardiomyocytes upregulates autophagic flux (2.6±1.4 fold) as compared to control HL1 cardiomyocytes. Moreover, HG treatment increased autophagic flux (1.4±0.25 fold) in HL1 cardiomyocytes, which was further increased by MMP9 ablation (1.7±0.24 fold) in HL1 cardiomyocytes. M‐mode echocardiography showed that percentage ejection fraction (% EF) was decreased in diabetic Akita as compared to WT (WT: 61.41±2.84 and Akita: 48.40±2.44) mice, but it was restored in DKO (61.28±0.71). There was no significant change in % EF in MMP9−⁄− (61.89±1.46) mice. These findings demonstrate a novel MMP9‐mediated regulation of autophagy in the diabetic heart, where ablation of MMP9 induces cardiac autophagic flux in the diabetic heart that may contribute to ameliorate cardiac dysfunction.