Novel Dual-Fluorescent Mitophagy Reporter Reveals a Reduced Mitophagy Flux in Type 1 Diabetic Mouse Heart

Abstract

Context: Patients with diabetes are susceptible to heart failure. Defective mitochondria can cause cardiac damage. Mitochondrial autophagy or mitophagy is a quality control mechanism that eliminates dysfunctional mitochondria through lysosome degradation. Mitophagy is essential for maintaining a pool of healthy mitochondria for normal cardiac function. However, the effect of diabetes on the functional status of cardiac mitophagy remains unclear. Objective: To determine and compare cardiac mitophagy flux between diabetic and nondiabetic mice. Methods: Using a novel dual fluorescent mitophagy reporter termed mt-Rosella, we labeled and traced mitochondrial fragments that are sequestered by the autophagosome and delivered to and degraded in the lysosome. Results: Mitophagic activity was reduced in high-glucose–treated cardiomyocytes and in the heart tissue of type 1 diabetic mice. Conclusions: Mitophagy was impaired in the heart of diabetic mice, suggesting that restoring or accelerating mitophagy flux may be a useful strategy to reduce cardiac injury caused by diabetes.

Mitochondrial Fission and Mitophagy Coordinately Restrict High Glucose Toxicity in Cardiomyocytes

“…Adult mouse cardiomyocytes (AMCs) were isolated from 8week-old transgenic mice that express the mitophagy reporter mt-Rosella in the heart (Kobayashi et al, 2020a) and cultured using the method described by Ackers-Johnson (Ackers-Johnson et al, 2016). Briefly, the heart was removed and perfused with collagenase solution by slow injection of the buffer into the left ventricle with a syringe.…”

“…Indeed, the mean mitochondrial particle size was substantially reduced by HG treatment (p < 0.01, n = 6). Alternatively, NRVCs were infected with adenovirus encoding the dual fluorescent mt-Rosella reporter which stains mitochondria and tracks their whereabouts in the cell as described below and previously (Catanzaro et al, 2019;Kobayashi et al, 2020a). These cells were fixed with 4% paraformaldehyde and imaged.…”

“…To directly visualize and quantify the mitochondrial fragments that are degraded through the mitophagic process, we constructed an adenovirus that encodes mt-Rosella, a novel mitophagy reporter (Catanzaro et al, 2019;Kobayashi et al, 2020a) that is a mitochondria-targeted dual-emission biosensor containing RFP and GFP as a fusion protein (Mijaljica et al, 2011). We infected NRVCs with Ad-mt-Rosella and compared mitophagy events (red fluorescent dots or puncta on the merged confocal images) in cells cultured under HG (30 mM) and NG (5.5 mM) conditions.…”

“…The protein levels of PINK1 and Parkin are decreased in the hearts of both type 1 and 2 diabetic animals (Xu et al, 2013;Tang et al, 2015;Shao et al, 2020), suggesting that mitophagy may be inhibited in the diabetic hearts. Using a novel dual fluorescent mitophagy reporter termed mt-Rosella, we labeled and traced mitochondrial fragments that are sequestered by the autophagosome and delivered to and degraded in the lysosome (Catanzaro et al, 2019;Kobayashi et al, 2020a). We found that mitophagic flux was indeed reduced in high glucose-treated cardiomyocytes and in the heart of streptozocin (STZ)-induced type 1 diabetic mice (Kobayashi et al, 2020a).…”

“…Using a novel dual fluorescent mitophagy reporter termed mt-Rosella, we labeled and traced mitochondrial fragments that are sequestered by the autophagosome and delivered to and degraded in the lysosome (Catanzaro et al, 2019;Kobayashi et al, 2020a). We found that mitophagic flux was indeed reduced in high glucose-treated cardiomyocytes and in the heart of streptozocin (STZ)-induced type 1 diabetic mice (Kobayashi et al, 2020a). Deletion of Parkin exacerbated diabetic cardiac injury in mice treated with STZ (Wang et al, 2019) or fed a high fat diet (Tong et al, 2019), suggesting that restoring Parkin-dependent mitophagy may be cardioprotective in diabetes.…”