Free
Original Contribution  |   July 2020
Novel Dual-Fluorescent Mitophagy Reporter Reveals a Reduced Mitophagy Flux in Type 1 Diabetic Mouse Heart
Author Notes
  • From the Department of Biomedical Sciences at the New York Institute of Technology College of Osteopathic Medicine in Old Westbury (Drs Kobayashi, Huang, and Liang and Mr Patel and Ms Kobayashi); and the Department of Endocrinology at the First Affiliated Hospital of Xi'an Jiaotong University in Xi’ an, China (Dr Zhao). Parts of this study were presented at the 77th and 79th Scientific Sessions of the American Diabetes Association June 2017, San Diego, CA, and June 2019, San Francisco, CA, respectively. 
  • Financial Disclosures: None reported. 
  • Support: This study was supported by a Career Development Grant (1-09-CD-09) from the American Diabetes Association. Dr Liang was supported by grants 1R15HL137130-01A1 and 1R15HL120027-01A1 from the National Institutes of Health. Dr Kobayashi was supported by the Scientist Development Grant (15SDG25080077) from the American Heart Association. 
  •  *Address correspondence to Qiangrong Liang, MD, PhD, New York Institute of Technology College of Osteopathic Medicine, Northern Blvd, PO Box 8000, Old Westbury, NY 11568-8000. Email: qliang03@nyit.edu
     
Article Information
Cardiovascular Disorders / Endocrinology / Diabetes
Original Contribution   |   July 2020
Novel Dual-Fluorescent Mitophagy Reporter Reveals a Reduced Mitophagy Flux in Type 1 Diabetic Mouse Heart
The Journal of the American Osteopathic Association, July 2020, Vol. 120, 446-455. doi:https://doi.org/10.7556/jaoa.2020.072
The Journal of the American Osteopathic Association, July 2020, Vol. 120, 446-455. doi:https://doi.org/10.7556/jaoa.2020.072
Web of Science® Times Cited: 1
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.

Diabetes is a major risk factor for the development of diabetic cardiomyopathy and heart failure, which are likely mediated by multiple mechanisms. Cardiomyocytes contain a large number of mitochondria, the powerhouses that provide most of the energy needed for heart contraction by producing adenosine triphosphatase through oxidative phosphorylation. Ironically, in diabetes, mitochondria are a predominant source of intracellular reactive oxygen species (ROS) and are themselves a primary target of oxidative injury.1,2 Damaged or otherwise dysfunctional mitochondria can further elevate ROS production through ROS-induced ROS release3,4 and may trigger leakage of prodeath factors, setting in motion a cycle that can induce cardiomyocyte death and heart failure.5-9 Thus, it is essential to repair or eliminate the injured mitochondria to maintain a pool of healthy mitochondria for normal cardiac function in patients with diabetes. An evolutionarily conserved endogenous cellular mechanism for ridding dysfunctional mitochondria is mitochondrial autophagy (mitophagy), a process that specifically sequesters mitochondria within the autophagosomes (termed mitophagosomes) and delivers them to the lysosomes, forming mitolysosomes for degradation and recycling. 
Mitochondrial injury has been consistently observed in the heart of patients with type 1 and type 2 diabetes mellitus and animal models, suggesting a failure of mitochondrial degradation by the mitophagy-lysosome pathway in diabetes.9-14 Studies have shown increased mitochondrial numbers and volume in the heart of diabetic animals, which was initially interpreted as being a result of enhanced mitochondrial biogenesis.11,14 However, these mitochondria are functionally defective, suggesting that they are more likely damaged mitochondria accumulated because of insufficient removal by mitophagy. In addition, the protein levels of PINK1 and parkin, 2 positive regulators of mitophagy, are decreased in the heart of diabetic animals,15,16 further supporting the possibility that there may be an insufficiency of mitophagy in the heart of diabetic animals that leads to an accumulation of injured mitochondria, contributing to the increased risk for heart failure. 
The approaches currently used for measuring mitophagy rely on the colocalization of a mitochondrial protein with a lysosomal or autophagosomal protein combined with surrogate biochemical assays. The occurrence of mitophagy can also be determined by transmission electron microscopy, which can provide direct evidence for autophagosomal engulfment of mitochondria. However, these approaches are relatively insensitive, cumbersome, and hard to quantify. A more powerful approach for measuring mitophagy is to visualize sequestered mitochondria in the autophagosome or mitophagosome and their subsequent delivery to the lysosomal compartment by using fluorescent probes. In this respect, 2 mitophagy reporter mice have been successfully used for determining mitophagy in mouse organs, including the heart, namely, mt-Keima (Keima-labeled mitochondria) and mCherry-green fluorescent protein (GFP)-mitochondrial fission 1 (FIS1) or mito-QC (quality control). Keima is a coral-derived acid-stable single-emission fluorescent protein that is directed to the mitochondrial matrix using the mitochondrial targeting sequence from cytochrome C oxidase subunit 8A (COX VIII). Keima emits a red fluorescent signal at acidic pH (586 nm) and a green fluorescent signal at higher pH (438 nm). Thus, mt-Keima that appear red are located and being degraded within the autolysosomes.17 Similarly, the fusion protein mCherry-GFP-FIS1 or mito-QC labels mitochondria by attaching to the mitochondrial outer membrane.18 This dual- emission pH biosensor consists of a pH-insensitive red fluorescent protein (RFP) connected to a pH-sensitive green fluorescent protein (GFP). Both emission signals are detected under neutral pH (mitochondria); however, the GFP signal is quenched under acidic pH (lysosomes). Thus, if the mito-QC-labeled mitochondria appear only red, they are located within the mitolysosomes waiting to be degraded.18 Both mt-Keima and mito-QC reporters have been used for monitoring the mitophagic process in the heart under several conditions. However, how diabetes affects mitophagy in the heart remains unclear. In the present study, we created and characterized another novel mitophagy reporter named mt-Rosella, which may be superior to the existing reporters in determining mitophagy flux in the heart. Using the mt-Rosella reporter, we investigated the functional status of mitophagy in cardiomyocytes exposed to high glucose (HG) and in the heart of type 1 diabetic mice. 
Methods
All animal protocols conformed to the Public Health Service Guide for Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the New York Institute of Technology College of Osteopathic Medicine. 
Adenoviral Vector Carrying the mt-Rosella Mitophagy Reporter
We constructed the replication-deficient adenovirus that encodes mt-Rosella.19,20 We tagged mt-Rosella with a mitochondrial targeting sequence from the gene that encodes the human COX VIII. The mt-Rosella coding sequence was subcloned into the pShuttle-CMV (cytomegalovirus) vector. Recombinant adenoviruses expressing mt-Rosella (Ad-mt-Rosella) were then generated and amplified using the AdEasy Adenoviral Vector System (Stratagene, 240009). 
Neonatal Rat Ventricular Cardiomyocyte Culture and HG Treatment
Neonatal rat ventricular cardiomyocytes (NRVCs) were isolated from 0- to 2-day-old Harlan Sprague-Dawley rats and cultured in Dulbecco Modified Eagle Medium (DMEM) as described previously.20 NRVCs were infected with Ad-mt-Rosella at a multiplicity of infection of 100 plaque-forming units for 18 hours and then cultured for 72 hours in glucose-free DMEM supplemented with 5.5 or 30 mM of glucose. The osmolarities of all media were made equal to 30 mM by adding different amounts of mannitol. All media contained 100 U/mL of penicillin and streptomycin. 
mt-Rosella Mitophagy Reporter Animals
To make transgenic mice expressing mt-Rosella in the heart, we cloned the mt-Rosella behind the cardiac α-myosin heavy chain (α-MHC) promoter. The linearized transgene was microinjected into a mouse embryo derived from the FVB/N strain of mice by Cyagen US Inc. The mt-Rosella mitophagy transgenic line was established and characterized. Diabetes was induced in 3-month-old mice by daily intraperitoneal injection of streptozotocin (STZ; 50 mg/kg body weight per day, dissolved in 10 mM sodium citrate buffer, pH 4.5) for 3 consecutive days. Control mice just received a citrate buffer. Blood samples were taken 1 week later for determining glucose concentrations. Mice with a fasting blood glucose level of 15 mM or greater were considered diabetic. 
Confocal Microscopy and Analysis of Mitophagy Events in NRVCs and Cardiac Tissues Using the mt-Rosella Mitophagy Reporter
Four hours before being euthanized, mt-Rosella mice were injected intraperitoneally with E64d and PepA each at 1 mg/kg for assessing the extent of mitochondrial accumulation in the lysosome. As a positive control for increased mitophagy flux, we injected some mice with carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 1 mg/kg body weight), a mitochondrial uncoupler commonly used to induce mitophagy in mice.25 Ad-mt-Rosella-infected NRVCs or cardiac tissue sections from the mt-Rosella mitophagy reporter mice were fixed with 4% paraformaldehyde prepared in phosphate-buffered saline for 10 minutes at room temperature. The slides were covered with coverslips and then observed with a laser scanning confocal microscope (OLYMPUS FV-1000). The confocal images were captured at ×600 magnification in 2 channels via 2 sequential excitations (458 nm, green, and 561 nm, red) using a 570- to 695-nm emission range. 
Dual-fluorescent images of NRVCs infected with Ad-mt-Rosella were split into red and green channels and contrast optimized. The green channel was subtracted from the red channel using ImageJ's Image Calculator feature, isolating the red-only portions of the original image. These red dots or puncta were mitophagy events that represented mitochondrial fragments trapped in the lysosome and waiting to be degraded. After optimization, each cell was individually outlined and analyzed. The analysis was performed with a size threshold of 1 µm2 to exclude small red noise particles and large nonspecific red particles that were unlikely to represent mitophagy events. Between 3 and 8 images (5-15 cells) were analyzed per treatment, and the mean numbers of red dots per cell from all treatments were compared. Similarly, confocal images from cardiac tissue sections were analyzed using ImageJ, and the mean numbers of red dots or mitophagy events from 3 fields per section were compared between animals from different treatment groups. For mitophagy flux analysis in cultured NRVCs, experiments were duplicated with the addition of lysosomal inhibitors pepstatin A (PepA, 12.5 ng/mL) and E64d (5 ng/mL), which were purchased from Research Products International and were dissolved in dimethyl sulfoxide. 
Statistical Analysis
Data were presented as mean (SD). A 2-way analysis of variance was used to analyze the differences between experimental groups followed by the Tukey multiple comparison test using GraphPad Prism software. The difference in mitophagy flux between control and HG or diabetic mice was analyzed using a t test. P<.01 was considered statistically significant. 
Results
Characterization of a Novel Mitophagy Reporter
To directly visualize and quantify the mitochondria that were degraded through the mitophagic process, we constructed an adenovirus that encoded mt-Rosella. As shown in Figure 1A, Rosella is a dual-emission biosensor comprising a pH-stable RFP linked to a pH-sensitive GFP.19 To target the RFP-GFP fusion protein to mitochondria, we tagged it with a mitochondrial targeting sequence from the gene that encodes human COX VIII. We infected NRVCs with Ad-mt-Rosella. As shown in Figure 1A, the mitochondria that appeared green or yellow on merged fluorescent images were located in the cytosolic compartments, while those that displayed only red fluorescence were being degraded within the lysosomes where the pH was low and the GFP was quenched. The red signal colocalized with mitochondrial protein TOM20 (Figure 1B), autophagosomal marker LC3 (microtubule-associated protein light chain 3, Figure 1C), and lysosome-associated membrane protein 1 (Figure 1D), confirming the specificity of the reporter. Treatment with ammonium chloride neutralized the acidic pH in the lysosome, which brought back the green fluorescence (Figure 1E), indicating the pH-dependent nature of the reporter. 
Figure 1.
Validation of the mt-Rosella mitophagy reporter. (A) Schematic of the mt-Rosella mitophagy reporter, which is composed of the mitochondrial target sequence, red fluorescent protein (RFP), and green fluorescent protein (GFP). Neonatal rat ventricular cardiomyocytes (NRVCs) were infected with Ad-mt-Rosella and immune-labeled with antibodies against (B) translocase of outer mitochondrial membrane 20 (TOM20), (C) light chain 3 (LC3), and (D) lysosomal-associated membrane protein 1 (LAMP1), and observed under a confocal microscope. The red dots on the overlaid images represent mitophagy events, indicating fragmented mitochondria that are trapped and being degraded in the lysosome. (E) Treatment with ammonium chloride (NH4Cl) brought back the green fluorescence, confirming the pH-dependent nature of the reporter.
Figure 1.
Validation of the mt-Rosella mitophagy reporter. (A) Schematic of the mt-Rosella mitophagy reporter, which is composed of the mitochondrial target sequence, red fluorescent protein (RFP), and green fluorescent protein (GFP). Neonatal rat ventricular cardiomyocytes (NRVCs) were infected with Ad-mt-Rosella and immune-labeled with antibodies against (B) translocase of outer mitochondrial membrane 20 (TOM20), (C) light chain 3 (LC3), and (D) lysosomal-associated membrane protein 1 (LAMP1), and observed under a confocal microscope. The red dots on the overlaid images represent mitophagy events, indicating fragmented mitochondria that are trapped and being degraded in the lysosome. (E) Treatment with ammonium chloride (NH4Cl) brought back the green fluorescence, confirming the pH-dependent nature of the reporter.
HG Inhibits Mitophagy Flux
Hyperglycemia is an independent risk factor for diabetic cardiac injury.21-24 To determine whether HG affected mitophagy, 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 normal glucose (NG; 5.5 mM) conditions. As shown in Figure 2, HG increased the number of red dots compared with NG, suggesting that HG might have enhanced mitophagy. However, mitophagy is a dynamic process, and its functional status cannot be determined simply by a snapshot of the number of mitochondrial fragments (red dots) present in the lysosome. Instead, mitophagy flux must be determined in the absence and presence of lysosomal degradation inhibitors to reveal the true dynamic changes of the mitophagic events or red dots during the whole degradation process. Thus, we treated cells with pepstatin A (PepA) and E64d, 2 lysosomal protease inhibitors that block lysosomal degradation, the last step of mitophagy. If a treatment enhances mitophagy, then a blockage of lysosomal degradation would cause further accumulation of the red dots on the merged confocal images. As shown in Figure 2, compared with DMSO control, PepA/E64d treatment led to a significant increase in the number of red dots in cells cultured in 5.5 mM of glucose, indicating an active mitophagy flux under an NG condition. However, PepA and E64d only resulted in a small increase in the number of red dots in cells cultured in 30 mM of glucose (HG, mean [SD], 9.2 [7.7] vs NG, 57.6 [20.3]; P<.01), suggesting that HG reduced the mitochondria degradation rate in the lysosome. In other words, HG inhibited mitophagy flux. This effect was not caused by increased osmolarity, since 24.5 mM of mannitol was added to the culture media containing 5.5 mM of glucose. 
Figure 2.
High glucose inhibits mitophagy flux in cardiomyocytes. Neonatal rat ventricular cardiomyocytes (NRVCs) were infected with adenoviruses expressing mt-Rosella (Ad-mt-Rosella) and cultured under normal (5.5 mM) or high glucose (high glucose, 30 mM) conditions for 72 hours. Confocal images were captured, mitophagy was analyzed using ImageJ and the number of mitophagy events or red dots per cell was calculated. Between 3 and 8 images (totaling between 5 and 15 cells) were captured per treatment over 3 separate experiments. For evaluating mitophagy flux, experiments were repeated with lysosomal inhibitors (pepstatin A [PepA] and E64d). Data were expressed as mean (SD) and were analyzed by 2-way analysis of variance (aP<.01 vs dimethyl sulfoxide (DMSO; n=4). Mitophagy flux (numbers on top of the bars) is defined as the difference of the numbers of red dots in the absence and presence of PepA and E64d, ie, the number of red dots represented by the open bar minus the number of red dots represented by the black bar. Data were expressed as mean (SD) and were analyzed by a t test (bP<.01 vs 5.5 mM glucose). Scale bar=20 µm.
Figure 2.
High glucose inhibits mitophagy flux in cardiomyocytes. Neonatal rat ventricular cardiomyocytes (NRVCs) were infected with adenoviruses expressing mt-Rosella (Ad-mt-Rosella) and cultured under normal (5.5 mM) or high glucose (high glucose, 30 mM) conditions for 72 hours. Confocal images were captured, mitophagy was analyzed using ImageJ and the number of mitophagy events or red dots per cell was calculated. Between 3 and 8 images (totaling between 5 and 15 cells) were captured per treatment over 3 separate experiments. For evaluating mitophagy flux, experiments were repeated with lysosomal inhibitors (pepstatin A [PepA] and E64d). Data were expressed as mean (SD) and were analyzed by 2-way analysis of variance (aP<.01 vs dimethyl sulfoxide (DMSO; n=4). Mitophagy flux (numbers on top of the bars) is defined as the difference of the numbers of red dots in the absence and presence of PepA and E64d, ie, the number of red dots represented by the open bar minus the number of red dots represented by the black bar. Data were expressed as mean (SD) and were analyzed by a t test (bP<.01 vs 5.5 mM glucose). Scale bar=20 µm.
Mitophagy Flux Is Reduced in the Diabetic Mouse Heart
To determine the effects of diabetes on mitophagy in the heart, we created transgenic mice that expressed mt-Rosella in the heart under the control of the α-MHC promoter. Diabetes was induced in 3-month old mt-Rosella mice by intraperitoneal STZ injections. Mitophagy flux was determined 9 weeks later when mice developed overt diabetic cardiac injury.15 Four hours before being euthanized, mt-Rosella mice were injected intraperitoneally with E64d and PepA each at 1 mg/kg for assessing the extent of mitochondrial accumulation in the lysosome. As a positive control for increased mitophagy flux, we injected some mice with carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 1 mg/kg body weight), a mitochondrial uncoupler commonly used to induce mitophagy in mice.25 CCCP was given for 3 consecutive days by intraperitoneal injections. Similar to our NRVC culture experiment, the red fluorescent dots in the merged confocal images represented mitochondrial fragments that were trapped within the lysosomes to be degraded (Figure 3). Very few red dots were present in the heart of nondiabetic mice, but they were easily seen in the heart of diabetic mice or mice treated with CCCP, suggesting that diabetes may have induced mitophagy, as did CCCP. Treatment with PepA and E64d led to a much greater increase in the number of red dots in the heart of CCCP-treated mice than in the heart of control mice, confirming the ability of CCCP to accelerate mitophagy flux. However, PepA and E64d only resulted in a smaller increase in the number of red dots in the heart of STZ-treated diabetic mice than in the heart of nondiabetic control mice, suggesting that diabetes reduced cardiac mitophagy flux. 
Figure 3.
Mitophagy flux is reduced in the heart of type 1 diabetic mice. Diabetes was induced in 3-month old mt-Rosella mice by intraperitoneal injections of streptozotocin (STZ). Mitophagy flux was determined 9 weeks later. Mice were injected intraperitoneal with PepA and E64d (each at 1 mg/kg) 4 hours before they were killed. As a positive control for increased mitophagy flux, some mice received carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 1 mg/kg) for 3 consecutive days by intraperitoneal injection. Confocal images from cardiac tissue sections were analyzed by using ImageJ and the mean numbers of red dots or mitophagy events from 3 fields per section were compared between animals from all groups. Data were expressed as mean (SD) and were analyzed by 2-way analysis of variance (*P<.01 vs DMSO control, n=4 for control and STZ, n=3 for CCCP). Mitophagy flux (numbers on top of the bars) is defined as the difference of the numbers of red dots in the absence and presence of PepA and E64d, ie, the number of red dots represented by the open bar minus the number of red dots represented by the black bar. Data were expressed as mean (SD) and were analyzed by a t test (#P<.01 vs nondiabetic control, n=4).
Figure 3.
Mitophagy flux is reduced in the heart of type 1 diabetic mice. Diabetes was induced in 3-month old mt-Rosella mice by intraperitoneal injections of streptozotocin (STZ). Mitophagy flux was determined 9 weeks later. Mice were injected intraperitoneal with PepA and E64d (each at 1 mg/kg) 4 hours before they were killed. As a positive control for increased mitophagy flux, some mice received carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 1 mg/kg) for 3 consecutive days by intraperitoneal injection. Confocal images from cardiac tissue sections were analyzed by using ImageJ and the mean numbers of red dots or mitophagy events from 3 fields per section were compared between animals from all groups. Data were expressed as mean (SD) and were analyzed by 2-way analysis of variance (*P<.01 vs DMSO control, n=4 for control and STZ, n=3 for CCCP). Mitophagy flux (numbers on top of the bars) is defined as the difference of the numbers of red dots in the absence and presence of PepA and E64d, ie, the number of red dots represented by the open bar minus the number of red dots represented by the black bar. Data were expressed as mean (SD) and were analyzed by a t test (#P<.01 vs nondiabetic control, n=4).
Discussion
Diabetes is a major risk factor for the development of various cardiovascular diseases, including atherosclerosis, hypertension, and diabetic cardiomyopathy. The lattermost is a heart muscle–specific disease independent of vascular pathology, for which there is no targeted therapy available, partially because of the limited understanding of the underlying mechanisms. In this respect, mitochondrial dysfunction and ROS have been suggested to play an important role in the pathogenesis of diabetic cardiomyopathy.1,2 If this were true, a potential therapeutic strategy for reducing diabetic cardiac injury would be to timely eliminate dysfunctional mitochondria, which could otherwise continuously generate ROS and release prodeath factors. Presumably, this could be achieved by improving the efficiency of mitophagy, a process that specifically degrades injured mitochondria through the autophagy-lysosome pathway. 
In our investigation of the functional status of mitophagy in NRVCs exposed to HG and in the heart of type 1 diabetic mice, we found that mitophagy flux was reduced in HG-treated NRVCs and in the heart of diabetic mice. Thus, restoring or accelerating mitophagy flux would be expected to facilitate the removal of defective mitochondria, thereby breaking the cycle of ROS generation and protecting the heart from diabetic injury. This hypothesis is currently being tested in our laboratory. 
Besides diabetes, dysregulated mitophagy has been linked to many other disease conditions, including aging and age-related diseases. There have been no convenient methods to analyze mitophagy, especially in vivo, until recently. Both mt-Keima and mito-QC reporters have been used for monitoring mitophagic processes in the heart under several conditions.17,18 However, our mt-Rosella mitophagy reporter may have several advantages over mt-Keima and mito-QC reporters. 
First, the mt-Rosella mitophagy reporter is under the control of the α-MHC promoter and is expressed only in cardiac myocytes. It is thus uniquely suited for monitoring mitophagy in these myocytes. In contrast, both mito-QC and mt-Keima are controlled by ubiquitous promoters and expressed in all types of cells in the heart. Investigation of mitophagy in cardiomyocytes may be interfered by signals from nonmyocytes in the heart if mito-QC and mt-Keima are used. 
Second, although mito-QC, mt-Keima, and mt-Rosella can all detect mitophagy events in the heart based on different pH levels in cytosol and lysosomes, mt-Rosella and mito-QC reporters may have 1 common advantage over mito-Keima: their versatility. When the Keima protein moves to the lysosome, it undergoes a gradual shift in fluorescence excitation, with an overlap in the emission spectra, which may complicate the interpretation of mitophagy because the distinction between acidic and neutral environments is not always obvious and likely influenced by tissue processing.17,18 Also, the Keima signal is lost upon conventional fixation, making it necessary to use freshly prepared sections for immediate visualization under a fluorescent microscope. In contrast, heart tissues from mt-Rosella and mito-QC mice can be fixed and saved for future visualization. Moreover, both mt-Rosella and mito-QC can be used to investigate not only mitophagy but also mitochondrial morphology and dynamics. 
The third potential advantage of mt-Rosella over mito-Keima is its usefulness in measuring mitophagy flux as shown in the present study. Although mito-Keima can provide a cumulative fluorescent readout of mitophagic activity for most upstream steps, including the formation of mitophagosomes and mitolysosomes, its usefulness in determining mitophagy flux at the last degradation step might be limited, since mito-Keima is believed to be resistant to acid proteases and thus may not be efficiently degraded in the lysosome.26 If so, PepA and E64d will not be able to differentiate between the increased formation of mitophagic vacuoles and the reduced degradation of them because the amount of mitochondrial fragments accumulated in the lysosome will remain the same either with or without lysosomal protease inhibitors. Apparently, this issue needs to be resolved by additional investigations. 
Last, mt-Rosella is located in the mitochondrial matrix and may more faithfully track the whole process of mitochondrial degradation as opposed to the mito-QC, which is targeted to the outer mitochondrial membrane and may lose track of mitochondrial fragments after the outer membrane has been degraded. 
Conclusion
We constructed a novel dual-fluorescent mitophagy reporter, mt-Rosella, which can label mitochondrial fragments that are sequestered by the autophagosome and delivered to and degraded in the lysosome. Using this reporter, we found that mitophagic activity was reduced in HG-treated NRVCs and in the heart of type 1 diabetic mice. Thus, approaches that accelerate mitophagy flux may be useful for reducing diabetic cardiac injury. 
Author Contributions
All authors provided substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; all authors drafted the article or revised it critically for important intellectual content; all authors gave final approval of the version of the article to be published; and all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. 
References
Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature. 2000;404(6779):787-790. doi: 10.1038/35008121 [CrossRef] [PubMed]
Kristal BS, Jackson CT, Chung HY, Matsuda M, Nguyen HD, Yu BP. Defects at center P underlie diabetes-associated mitochondrial dysfunction. Free Radic Biol Med. 1997;22(5):823-833. doi: 10.1016/s0891-5849(96)00428-5 [CrossRef] [PubMed]
Brady NR, Hamacher-Brady A, Westerhoff HV, Gottlieb RA. A wave of reactive oxygen species (ROS)-induced ROS release in a sea of excitable mitochondria. Antioxid Redox Signal. 2006;8(9-10):1651-1665. doi: 10.1089/ars.2006.8.1651 [CrossRef] [PubMed]
Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial ROS-induced ROS release: an update and review. Biochim Biophys Acta. 2006;1757(5-6):509-517. doi: 10.1016/j.bbabio.2006.04.029
Cai L, Li W, Wang G, Guo L, Jiang Y, Kang YJ. Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway. Diabetes. 2002;51(6):1938-1948. doi: 10.2337/diabetes.51.6.1938 [CrossRef] [PubMed]
Ghosh S, Pulinilkunnil T, Yuen G, et al. Cardiomyocyte apoptosis induced by short-term diabetes requires mitochondrial GSH depletion. Am J Physiol Heart Circ Physiol. 2005;289(2):H768-H776. doi: 10.1152/ajpheart.00038.2005 [CrossRef] [PubMed]
Malhotra A, Begley R, Kang BP, et al. PKC-{epsilon}-dependent survival signals in diabetic hearts. Am J Physiol Heart Circ Physiol. 2005;289(4):H1343-H1350. doi: 10.1152/ajpheart.01200.2004 [CrossRef] [PubMed]
Shen X, Zheng S, Metreveli NS, Epstein PN. Protection of cardiac mitochondria by overexpression of MnSOD reduces diabetic cardiomyopathy. Diabetes. 2006;55(3):798-805. doi: 10.2337/diabetes.55.03.06.db05-1039 [CrossRef] [PubMed]
Frustaci A, Kajstura J, Chimenti C, et al. Myocardial cell death in human diabetes. Circ Res. 2000;87(12):1123-1132. doi: 10.1161/01.res.87.12.1123 [CrossRef] [PubMed]
Tomita M, Mukae S, Geshi E, Umetsu K, Nakatani M, Katagiri T. Mitochondrial respiratory impairment in streptozotocin-induced diabetic rat heart. Jpn Circ J. 1996;60(9):673-682. doi: 10.1253/jcj.60.673 [CrossRef] [PubMed]
Shen X, Zheng S, Thongboonkerd V, et al. Cardiac mitochondrial damage and biogenesis in a chronic model of type 1 diabetes. Am J Physiol Endocrinol Metab. 2004;287(5):e896-e905. doi: 10.1152/ajpendo.00047.2004 [CrossRef] [PubMed]
Shen X, Ye G, Metreveli NS, Epstein PN. Cardiomyocyte defects in diabetic models and protection with cardiac-targeted transgenes. Methods Mol Med. 2005;112:379-388. doi: 10.1385/1-59259-879-x:379 [PubMed]
Boudina S, Abel ED. Mitochondrial uncoupling: a key contributor to reduced cardiac efficiency in diabetes. Physiology (Bethesda. ). 2006;21:250-258. doi: 10.1152/physiol.00008.2006 [PubMed]
Bugger H, Abel ED. Molecular mechanisms for myocardial mitochondrial dysfunction in the metabolic syndrome. Clin Sci (Lond. ). 2008;114(3):195-210. doi: 10.1042/CS20070166 [CrossRef] [PubMed]
Xu X, Kobayashi S, Chen K, et al. Diminished autophagy limits cardiac injury in mouse models of type 1 diabetes. J Biol Chem. 2013;288(25):18077-18092. doi: 10.1074/jbc.M113.474650 [CrossRef] [PubMed]
Tang Y, Liu J, Long J. Phosphatase and tensin homolog-induced putative kinase 1 and Parkin in diabetic heart: role of mitophagy. J Diabetes Investig. 2015;6(3):250-255. doi: 10.1111/jdi.12302 [CrossRef] [PubMed]
Sun N, Yun J, Liu J, et al. Measuring In Vivo Mitophagy. Mol Cell. 2015;60(4):685-696. doi: 10.1016/j.molcel.2015.10.009 [CrossRef] [PubMed]
McWilliams TG, Prescott AR, Allen GF, et al.  . mito-QC illuminates mitophagy and mitochondrial architecture in vivo. J Cell Biol. 2016;214(3):333-345. doi: 10.1083/jcb.201603039 [CrossRef] [PubMed]
Mijaljica D, Prescott M, Devenish RJ. A fluorescence microscopy assay for monitoring mitophagy in the yeast Saccharomyces cerevisiae. J Vis Exp. 2011;(53):2779. doi: 10.3791/2779
Kobayashi S, Xu X, Chen K, Liang Q. Suppression of autophagy is protective in high glucose-induced cardiomyocyte injury. Autophagy. 2012;8(4):577-592. doi: 10.4161/auto.18980 [CrossRef] [PubMed]
Stratton IM, Adler AI, Neil HA, et al. Association of glycaemia with macrovascular and microvascular complications of type 2 diabetes (UKPDS 35): prospective observational study. BMJ. 2000;321(7258):405-412. doi: 10.1136/bmj.321.7258.405
Boudina S, Abel ED. Diabetic cardiomyopathy revisited. Circulation. 2007;115(25):3213-3223. doi: 10.1161/CIRCULATIONAHA.106.679597 [CrossRef] [PubMed]
Poornima IG, Parikh P, Shannon RP. Diabetic cardiomyopathy: the search for a unifying hypothesis. Circ Res. 2006;98(5):596-605. doi: 10.1161/01.RES.0000207406.94146.c2 [CrossRef] [PubMed]
Iribarren C, Karter AJ, Go AS, Liu JY, Sidney S, Selby JV. Glycemic control and heart failure among adult patients with diabetes. Circulation. 2001;103(22):2668-2673. doi: 10.1161/01.cir.103.22.2668 [CrossRef] [PubMed]
Hoshino A, Mita Y, Okawa Y, et al. Cytosolic p53 inhibits Parkin- mediated mitophagy and promotes mitochondrial dysfunction in the mouse heart. Nat Commun. 2013;4:2308. doi: 10.1038/ncomms3308 [CrossRef] [PubMed]
Katayama H, Kogure T, Mizushima N, Yoshimori T, Miyawaki A. A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem Biol. 2011;18(8):1042-1052. doi: 10.1016/j.chembiol.2011.05.013 [CrossRef] [PubMed]
Figure 1.
Validation of the mt-Rosella mitophagy reporter. (A) Schematic of the mt-Rosella mitophagy reporter, which is composed of the mitochondrial target sequence, red fluorescent protein (RFP), and green fluorescent protein (GFP). Neonatal rat ventricular cardiomyocytes (NRVCs) were infected with Ad-mt-Rosella and immune-labeled with antibodies against (B) translocase of outer mitochondrial membrane 20 (TOM20), (C) light chain 3 (LC3), and (D) lysosomal-associated membrane protein 1 (LAMP1), and observed under a confocal microscope. The red dots on the overlaid images represent mitophagy events, indicating fragmented mitochondria that are trapped and being degraded in the lysosome. (E) Treatment with ammonium chloride (NH4Cl) brought back the green fluorescence, confirming the pH-dependent nature of the reporter.
Figure 1.
Validation of the mt-Rosella mitophagy reporter. (A) Schematic of the mt-Rosella mitophagy reporter, which is composed of the mitochondrial target sequence, red fluorescent protein (RFP), and green fluorescent protein (GFP). Neonatal rat ventricular cardiomyocytes (NRVCs) were infected with Ad-mt-Rosella and immune-labeled with antibodies against (B) translocase of outer mitochondrial membrane 20 (TOM20), (C) light chain 3 (LC3), and (D) lysosomal-associated membrane protein 1 (LAMP1), and observed under a confocal microscope. The red dots on the overlaid images represent mitophagy events, indicating fragmented mitochondria that are trapped and being degraded in the lysosome. (E) Treatment with ammonium chloride (NH4Cl) brought back the green fluorescence, confirming the pH-dependent nature of the reporter.
Figure 2.
High glucose inhibits mitophagy flux in cardiomyocytes. Neonatal rat ventricular cardiomyocytes (NRVCs) were infected with adenoviruses expressing mt-Rosella (Ad-mt-Rosella) and cultured under normal (5.5 mM) or high glucose (high glucose, 30 mM) conditions for 72 hours. Confocal images were captured, mitophagy was analyzed using ImageJ and the number of mitophagy events or red dots per cell was calculated. Between 3 and 8 images (totaling between 5 and 15 cells) were captured per treatment over 3 separate experiments. For evaluating mitophagy flux, experiments were repeated with lysosomal inhibitors (pepstatin A [PepA] and E64d). Data were expressed as mean (SD) and were analyzed by 2-way analysis of variance (aP<.01 vs dimethyl sulfoxide (DMSO; n=4). Mitophagy flux (numbers on top of the bars) is defined as the difference of the numbers of red dots in the absence and presence of PepA and E64d, ie, the number of red dots represented by the open bar minus the number of red dots represented by the black bar. Data were expressed as mean (SD) and were analyzed by a t test (bP<.01 vs 5.5 mM glucose). Scale bar=20 µm.
Figure 2.
High glucose inhibits mitophagy flux in cardiomyocytes. Neonatal rat ventricular cardiomyocytes (NRVCs) were infected with adenoviruses expressing mt-Rosella (Ad-mt-Rosella) and cultured under normal (5.5 mM) or high glucose (high glucose, 30 mM) conditions for 72 hours. Confocal images were captured, mitophagy was analyzed using ImageJ and the number of mitophagy events or red dots per cell was calculated. Between 3 and 8 images (totaling between 5 and 15 cells) were captured per treatment over 3 separate experiments. For evaluating mitophagy flux, experiments were repeated with lysosomal inhibitors (pepstatin A [PepA] and E64d). Data were expressed as mean (SD) and were analyzed by 2-way analysis of variance (aP<.01 vs dimethyl sulfoxide (DMSO; n=4). Mitophagy flux (numbers on top of the bars) is defined as the difference of the numbers of red dots in the absence and presence of PepA and E64d, ie, the number of red dots represented by the open bar minus the number of red dots represented by the black bar. Data were expressed as mean (SD) and were analyzed by a t test (bP<.01 vs 5.5 mM glucose). Scale bar=20 µm.
Figure 3.
Mitophagy flux is reduced in the heart of type 1 diabetic mice. Diabetes was induced in 3-month old mt-Rosella mice by intraperitoneal injections of streptozotocin (STZ). Mitophagy flux was determined 9 weeks later. Mice were injected intraperitoneal with PepA and E64d (each at 1 mg/kg) 4 hours before they were killed. As a positive control for increased mitophagy flux, some mice received carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 1 mg/kg) for 3 consecutive days by intraperitoneal injection. Confocal images from cardiac tissue sections were analyzed by using ImageJ and the mean numbers of red dots or mitophagy events from 3 fields per section were compared between animals from all groups. Data were expressed as mean (SD) and were analyzed by 2-way analysis of variance (*P<.01 vs DMSO control, n=4 for control and STZ, n=3 for CCCP). Mitophagy flux (numbers on top of the bars) is defined as the difference of the numbers of red dots in the absence and presence of PepA and E64d, ie, the number of red dots represented by the open bar minus the number of red dots represented by the black bar. Data were expressed as mean (SD) and were analyzed by a t test (#P<.01 vs nondiabetic control, n=4).
Figure 3.
Mitophagy flux is reduced in the heart of type 1 diabetic mice. Diabetes was induced in 3-month old mt-Rosella mice by intraperitoneal injections of streptozotocin (STZ). Mitophagy flux was determined 9 weeks later. Mice were injected intraperitoneal with PepA and E64d (each at 1 mg/kg) 4 hours before they were killed. As a positive control for increased mitophagy flux, some mice received carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 1 mg/kg) for 3 consecutive days by intraperitoneal injection. Confocal images from cardiac tissue sections were analyzed by using ImageJ and the mean numbers of red dots or mitophagy events from 3 fields per section were compared between animals from all groups. Data were expressed as mean (SD) and were analyzed by 2-way analysis of variance (*P<.01 vs DMSO control, n=4 for control and STZ, n=3 for CCCP). Mitophagy flux (numbers on top of the bars) is defined as the difference of the numbers of red dots in the absence and presence of PepA and E64d, ie, the number of red dots represented by the open bar minus the number of red dots represented by the black bar. Data were expressed as mean (SD) and were analyzed by a t test (#P<.01 vs nondiabetic control, n=4).