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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., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2020 Wiley Periodicals LLC.)

Mitochondrial respiration through electron transport chain (ETC) activity generates ATP and reactive oxygen species in eukaryotic cells. The modulation of mitochondrial respiration in vivo or under physiological conditions remains elusive largely due to the lack of appropriate approach to monitor ETC activity in a real-time manner. Here, we show that ETC-coupled mitochondrial flash is a novel biomarker for monitoring mitochondrial respiration under pathophysiological conditions in cultured adult cardiac myocyte and perfused beating heart. Through real-time confocal imaging, we follow the frequency of a transient bursting fluorescent signal, named mitochondrial flash, from individual mitochondria within intact cells expressing a mitochondrial matrix-targeted probe, mt-cpYFP (mitochondrial-circularly permuted yellow fluorescent protein). This mt-cpYFP recorded mitochondrial flash has been shown to be composed of a major superoxide signal with a minor alkalization signal within the mitochondrial matrix. Through manipulating physiological substrates for mitochondrial respiration, we find a close coupling between flash frequency and the ETC electron flow, as measured by oxygen consumption rate in cardiac myocyte. Stimulating electron flow under physiological conditions increases flash frequency. On the other hand, partially block or slowdown electron flow by inhibiting the F0F1 ATPase, which represents a pathological condition, transiently increases then decreases flash frequency. Limiting electron entrance at complex I by knocking out Ndufs4, an assembling subunit of complex I, suppresses mitochondrial flash activity. These results suggest that mitochondrial electron flow can be monitored by real-time imaging of mitochondrial flash. The mitochondrial flash frequency could be used as a novel biomarker for mitochondrial respiration under physiological and pathological conditions., (Copyright © 2015 the American Physiological Society.)

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 °C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Superoxide flashes are transient bursts of superoxide production within the mitochondrial matrix that are detected using the superoxide-sensitive biosensor, mitochondria-targeted circularly permuted YFP (mt-cpYFP). However, due to the pH sensitivity of mt-cpYFP, flashes were suggested to reflect transient events of mitochondrial alkalinization. Here, we simultaneously monitored flashes with mt-cpYFP and mitochondrial pH with carboxy-SNARF-1. In intact cardiac myocytes and purified skeletal muscle mitochondria, robust mt-cpYFP flashes were accompanied by only a modest increase in SNARF-1 ratio (corresponding to a pH increase of <0.1), indicating that matrix alkalinization is minimal during an mt-cpYFP flash. Individual flashes were also accompanied by stepwise increases of MitoSOX signal and decreases of NADH autofluorescence, supporting the superoxide origin of mt-cpYFP flashes. Transient matrix alkalinization induced by NH4Cl only minimally influenced flash frequency and failed to alter flash amplitude. However, matrix acidification modulated superoxide flash frequency in a bimodal manner. Low concentrations of nigericin (< 100 nM) that resulted in a mild dissipation of the mitochondrial pH gradient increased flash frequency, whereas a maximal concentration of nigericin (5 μm) collapsed the pH gradient and abolished flash activity. These results indicate that mt-cpYFP flash events reflect a burst in electron transport chain-dependent superoxide production that is coincident with a modest increase in matrix pH. Furthermore, flash activity depends strongly on a combination of mitochondrial oxidation and pH gradient.