visualizing-mitophagy-with-fluorescent-dyes-for-mitochondria

Visualizing Mitophagy with Fluorescent Dyes for Mitochondria and Lysosome

Mitochondria, being the powerhouses of the cell, play important roles in bioenergetics, free radical generation, calcium homeostasis, and apoptosis. ...

Tongji University

Wenzhou Medical University

Mitochondria, the powerhouse of eukaryotic cells, are highly dynamic organelle. Mitochondrial fission, fusion, kissing and contraction have been reported over and over again in non-static cells, such as fibroblast, with tubular mitochondrial networks. Even though the fluorescence propagation among mitochondria of mature cardiomyocytes had been captured using mitochondrial matrix targeted photoactivatable GFP (PAGFP) or MitoDendra proteins, there are no direct evidence that single real time mitochondrial dynamics events exist in mature cardiomyocytes with ball-like mitochondria. Here we first time revealed the visualizable single mitochondrial dynamics events in adult mature cardiomyocytes by the mitochondrial flash (mitoflash). We found fission, fusion, contraction and kissing were accompanied by a mitoflash event. Metabolism could increase mitochondrial contraction. Fusion and Kissing mediated inter-mitochondrial communication with higher frequency than fission. These results demonstrate that mitochondria of static mature cardiomyocytes are undergoing the rare, but real dynamics change.

Mitoflash lights single mitochondrial dynamics events in mature cardiomyocytes.

Cultured adult mouse and rat cardiomyocytes are the best and low-cost cell model for cardiac cellular physiology, pathology, drug toxicity screening, and intervention. The functions of mouse cardiomyocytes decline faster than rat cardiomyocytes in culture conditions. However, little is known about the difference of mitochondrial function between cultured mouse and rat myocytes.

Comparative study on isolation and mitochondrial function of adult mouse and rat cardiomyocytes.

Mitochondrial fusion and fission are essential for heart function. Abrogating mitochondrial dynamism leads to cardiomyopathy. Excessive mitochondrial fragmentation is involved in most heart diseases, thus enhancing mitochondrial fusion will be a potential therapeutic strategy. To understand the effects of promoting mitochondrial fusion in adult cardiac, we investigated mice hearts, and cultured murine embryonic fibroblasts (MEFs), in which mitofusin 2 (Mfn2) overexpressed or dynamin-related protein 1 (Drp1) was abrogated concomitantly forcing mitochondrial fusion. Parallel studies revealed that fission-defective Drp1 knockout hearts and MEFs evoked stronger mitochondrial enlargement, enhanced mitophagy with mitochondrial volume decrease and increased mitochondrial calcium uptake, superoxide production, and permeability transition pore opening, contributed to cardiomyocyte apoptosis and dilated cardiomyopathy. Mfn2 overexpression in the adult heart is comparable with the control except for slight mitochondrial enlargement and mitochondrial volume increase, but without mitophagy induction. Moreover, Mfn2 overexpression increases mitochondrial biogenesis and fusion could protect against mitochondrial fragmentation and Drp1 deletion evoking mitophagy in MEFs. Our findings indicate that mitochondrial fusion provoked by fusion promotion and fission inhibition direct the different fate of heart, Mfn2 upregulation other than Drp1 downregulation well maintains heart mitochondrial function is a more safe strategy for correcting excessive mitochondrial fragmentation in hearts.

Mitochondrial fusion mediated by fusion promotion and fission inhibition directs adult mouse heart function toward a different direction.

The previous research has shown that mitochondrial flash (mitoflash) genesis are functionally and mechanistically integrated with mitochondrial electron transport chain (ETC) energy metabolism. However, the response of mitoflash to superoxide is not entirely consistent with the response of MitoSOX Red. The generation mechanism of mitoflash is still unclear. Here, we investigated mitoflash activities, using the different combinations of ETC substrates and inhibitors, in permeabilized cardiomyocytes or hearts. We found that blocking the complete electron flow, from Complex I to IV, with any one of ETC inhibitors including rotenone (Rot), antimycin A (AntA), myxothiazol (Myxo), stigmatellin, and sodium cyanide, will lead to the abolishment of mitoflashes triggered by substrates in adult permeabilized cardiomyocytes. However, Myxo boosted mitoflashes triggered by the reverse electron of N,N,N',N'-tetramethyl-p-phenylenediamine/ascorbate. Moreover, Rot and AntA furtherly enhanced mitoflash activity rather than depressed it, suggesting that mitoflashes generated at the Complex III Qo site. Meanwhile, the inhibition of Complex III protein expression resulted in the activity of Complex III decrease, which decreased mitoflash frequency. The function defect (no change of protein level) of the Qo site of Complex III in aging hearts augmented mitoflash generation confirmed the Qo site function was critical to mitoflash genesis. Thus, our results indicate that mitoflash detected by circularly permuted yellow fluorescent protein is generated at the Qo site of Complex III.

Mitoflash generated at the Qo site of mitochondrial Complex III.

Isolation of high-quantity and high-quality ventricular cardiomyocytes from adult rats is critical to study heart physiology and pathology and for drug toxicity screening. It remains challenging to produce a high yield of viable cardiomyocytes from rats. Here, we present our modified enzymatic digestion protocol that relies on the Langendorff device to generate large numbers of viable cardiomyocytes consistently. The most critical parts of this protocol are the selection of rat age and digestion time to obtain viable cardiomyocytes. For complete details on the use and execution of this protocol, please refer to Liu et al. (2019) and Qin et al. (2020).

Protocol for Isolation of Viable Adult Rat Cardiomyocytes with High Yield.

Analysis of mitochondrial respiration function represented by the oxygen consumption rate is necessary for assessing mitochondrial respiration function. This protocol describes steps to evaluate the respiration function of mitochondria  in saponin-permeabilized cardiomyocytes. In permeabilized cells, mitochondria are in a relatively integrated cellular system, and mitochondrial respiration is more physiologically relevant than isolated mitochondria. For complete details on the use and execution of this protocol, please refer to Gong et al. (2015a) and Gong et al. (2015b).

Protocol for Measurement of Oxygen Consumption Rate  in Permeabilized Cardiomyocytes.

We describe a protocol for imaging a mitochondrial fluorescence transient increase event (Mitoflash) in live cardiomyocytes using a confocal microscope. Mitoflash, detected by mitochondria-targeted circularly permuted fluorescent protein (mt-cpYFP), can be used to assess mitochondrial respiration function . The protocol is also suitable for live-cell imaging of other adherent cells, including fibroblasts and hepatocytes. For complete details on the use and execution of this protocol, please refer to Gong et al. (2014) and Gong et al. (2015).

Protocol for Imaging of Mitoflashes in Live Cardiomyocytes.

Here, we present a modified protocol for a mouse heart failure (HF) model using minimally invasive transverse aortic constriction (miTAC). miTAC is a more effective method in mice than the standard open-chest transverse aortic constriction (TAC) to generate an HF model. miTAC does not require the cutting of the ribs or tracheal intubation with artificial ventilation; it also has a higher survival rate. The successful outcome of the HF model can be verified using transthoracic echocardiography and histology. For complete details on the use and execution of this protocol, please refer to Hu et al. (2003) and Richards et al. (2019).

Modified Protocol for A Mouse Heart Failure Model Using Minimally Invasive Transverse Aortic Constriction.

Mitofusin 2 (Mfn2) is a transmembrane GTPase located on the mitochondrial outer membrane that contributes to mitochondrial network regulation. It is an essential multifunctional protein that participates in various biological processes under physical and pathological conditions, including mitochondrial fusion, reticulum-mitochondria contacts, mitochondrial quality control, and apoptosis. Mfn2 dysfunctions have been found to contribute to cardiovascular diseases, such as ischemia-reperfusion injury, heart failure, and dilated cardiomyopathy. Here, this review mainly focuses on what is known about the structure and function of Mfn2 and its crucial role in heart failure.

Mitochondrial Fusion Protein Mfn2 and Its Role in Heart Failure.

Mitochondrial pH is a vital parameter of the mitochondrial environment, which determines the rate of many mitochondrial functions, including metabolism, membrane potential, fate, etc. Abnormal mitochondrial pH is always closely related to the health status of cells. Analyzing mitochondrial pH can serve as a proxy for mitochondrial and cellular function. This protocol describes the use of SNARF-1 AM, a pH-sensitive fluorophore, to measure mitochondrial pH. This protocol details the steps to evaluate mitochondrial pH in live adult cardiomyocytes using confocal microscopy. The protocol can be adapted to other adherent cell types. For complete details on the use and execution of this protocol, please refer to Wei-LaPierre et al. (2013).

Calibration and measurement of mitochondrial pH in intact adult rat cardiomyocytes.

Letter to the editor: Compromised hepatic mitochondrial fatty acid oxidation and reduced markers of mitochondrial turnover in human NAFLD.

Mitochondria are highly dynamic organelles that participate in ATP generation and involve calcium homeostasis, oxidative stress response, and apoptosis. Dysfunctional or damaged mitochondria could cause serious consequences even lead to cell death. Therefore, maintaining the homeostasis of mitochondria is critical for cellular functions. Mitophagy is a process of selectively degrading damaged mitochondria under mitochondrial toxicity conditions, which plays an essential role in mitochondrial quality control. The abnormal mitophagy that aggravates mitochondrial dysfunction is closely related to the pathogenesis of many diseases. As the myocardium is a highly oxidative metabolic tissue, mitochondria play a central role in maintaining optimal performance of the heart. Dysfunctional mitochondria accumulation is involved in the pathophysiology of cardiovascular diseases, such as myocardial infarction, cardiomyopathy and heart failure. This review discusses the most recent progress on mitophagy and its role in cardiovascular disease.

Mitochondrial autophagy: molecular mechanisms and implications for cardiovascular disease.

Mitochondria with structural and functional integrity are essential for maintaining mitochondrial function and cardiac homeostasis. It is involved in the pathogenesis of many diseases. Peroxisome proliferator-activated receptor  coactivator 1  (PGC-1), acted as a transcriptional cofactor, is abundant in the heart, which modulates mitochondrial biogenesis and mitochondrial dynamics and mitophagy to sustain a steady-state of mitochondria. Cumulative evidence suggests that dysregulation of PGC-1 is closely related to the onset and progression of heart failure. PGC-1 deficient-mice can lead to worse cardiac function under pressure overload compared to sham. Here, this review mainly focuses on what is known about its regulation in mitochondrial functions, as well as its crucial role in heart failure.

PGC-1-Mediated Mitochondrial Quality Control: Molecular Mechanisms and Implications for Heart Failure.

The redox state is a crucial determinant of the maturation transition of cardiomyocytes in vivo. Mitochondria, the primary site of superoxide generation, are very sensitive to various stimulations, including oxygen and nutrient supply. How mitochondrial superoxide affects the differentiation and development of induced pluripotent stem cell (iPSC)-derived cardiac myocytes (iPS-CMs) is not completely clear. To address the questions, we monitored the superoxide level during the differentiation and development of human iPS-CMs using MitoSOX. Mitochondria-targeted antioxidant Mito-TEMPO was used to treat hiPS-CMs in the differentiation period. We found that mitochondrial superoxide generation was dramatically enhanced during the differentiation and early development of iPS-CMs. Increased oxidative stress induced oxidative damage to macromolecules in iPS-CMs, such as lipids, proteins, and DNA. Mito-TEMPO protected mitochondrial functions, alleviated oxidative damage to lipids, proteins, and DNA and improved cellular structure and fatty acid utilization. Our findings confirmed that iPS-CM suffered from oxidative stress during differentiation and that mitochondrial-targeted antioxidant is beneficial for the maturation of iPS-CMs.

Inhibition of mitochondrial superoxide promotes the development of hiPS-CMs during differentiation.

A novel marine Gram-stain-negative, aerobic, rod-shaped bacterium, designated as strain PS1, was isolated from the deep-sea sediments of the Mariana Trench and characterized phylogenetically and phenotypically. Bacterial optimal growth occurred at 35 °C (ranging 10-45 °C), pH 6 (ranging pH 5-10) and with 11% (w/v) NaCl (ranging 0-17%). The 16S rRNA gene sequence similarity results revealed that strain PS1 was most closely related to Pseudomonas stutzeri ATCC 17588, Pseudomonas nitrititolerans GL14, Pseudomonas zhaodongensis NEAU-ST5-21, Pseudomonas xanthomarina DSM 18231 and Pseudomonas kunmingensis HL22-2 with 98.3-98.7%. The digital DNA-DNA hybridization values and the average nucleotide identity between strain PS1 and the reference strains were 20.4-40.1% and 78.7-79.4%, respectively. The major respiratory quinone is ubiquinone Q-9. The major polar lipids were phosphatidylethanolamine, diphosphatidyglycerol, phosphatidylglycerol, phosphatidylcholine, aminoglycolipid, two unidentified glycolipids and one unidentified lipid. The predominant cellular fatty acids of strain PS1 were summed feature 8 (Cω7c and/or Cω6c), summed feature 3 (Cω7c and/or Cω6c), C and cyclo-C ω8c. The G + C content of the genomic DNA was 63.0%. The combined genotypic and phenotypic data indicated that strain PS1 represents a novel species of the genus Pseudomonas, for which the name Pseudomonas marianensis sp. nov. is proposed, with the type strain PS1 (= DSM 112238 = MCCC 1K05112).

Article	Year
Mitoflash lights single mitochondrial dynamics events in mature cardiomyocytes. Biochemical and biophysical research communications\| PubMed ID: 29928879	2018
Comparative study on isolation and mitochondrial function of adult mouse and rat cardiomyocytes. Journal of molecular and cellular cardiology\| PubMed ID: 31521710	2019
Mitochondrial fusion mediated by fusion promotion and fission inhibition directs adult mouse heart function toward a different direction. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 31914595	2020
Mitoflash generated at the Qo site of mitochondrial Complex III. Journal of cellular physiology\| PubMed ID: 32930405	2021
Protocol for Isolation of Viable Adult Rat Cardiomyocytes with High Yield. STAR protocols\| PubMed ID: 33111091	2020
Protocol for Measurement of Oxygen Consumption Rate in Permeabilized Cardiomyocytes. STAR protocols\| PubMed ID: 33111108	2020
Protocol for Imaging of Mitoflashes in Live Cardiomyocytes. STAR protocols\| PubMed ID: 33111128	2020
Modified Protocol for A Mouse Heart Failure Model Using Minimally Invasive Transverse Aortic Constriction. STAR protocols\| PubMed ID: 33377080	2020
Mitochondrial Fusion Protein Mfn2 and Its Role in Heart Failure. Frontiers in molecular biosciences\| PubMed ID: 34026850	2021
Calibration and measurement of mitochondrial pH in intact adult rat cardiomyocytes. STAR protocols\| PubMed ID: 34036286	2021
Letter to the editor: Compromised hepatic mitochondrial fatty acid oxidation and reduced markers of mitochondrial turnover in human NAFLD. Hepatology (Baltimore, Md.)\| PubMed ID: 35491446	2022
Mitochondrial autophagy: molecular mechanisms and implications for cardiovascular disease. Cell death & disease\| PubMed ID: 35534453	2022
PGC-1-Mediated Mitochondrial Quality Control: Molecular Mechanisms and Implications for Heart Failure. Frontiers in cell and developmental biology\| PubMed ID: 35721484	2022
Inhibition of mitochondrial superoxide promotes the development of hiPS-CMs during differentiation. Free radical biology & medicine\| PubMed ID: 35952922	2022
Pseudomonas marianensis sp. nov., a marine bacterium isolated from deep-sea sediments of the Mariana Trench. Archives of microbiology\| PubMed ID: 36131209	2022

Bilin Liu

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