Perfused murine heart optical transmission spectroscopy using optical catheter and integrating sphere: Effects of ischemia/reperfusion

Bauer, Theodore J.; Giles, Abigail; Sun, Junhui; Femnou, Armel N.; Covián, Raúl; Murphy, Elizabeth; Balaban, Robert S.

doi:10.1016/j.ab.2019.113443

Cited by 12 publications

(5 citation statements)

References 36 publications

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“…Transmural absorbance spectroscopy is a robust method for monitoring cardiac chromophores in the intact heart (Bauer et al., 2019; Femnou et al., 2017; Giles et al., 2018; Kuzmiak‐Glancy et al., 2018). In addition to mitochondrial chromophores and OxyMb and DeoxyMb, these studies evaluated the ability of optical spectroscopy to monitor MetMb, enabling a kinetic evaluation of the Mb/MbR system in the intact heart.…”

Section: Resultsmentioning

confidence: 99%

“…Spectral fitting was performed using chromophore reference standards and a previously described LabVIEW-based program. Cytochromes c and c 1 , as well as Complex III haem b H and b L reference spectra were identical to previous publications (Bauer et al, 2019;Femnou et al, 2017;Giles et al, 2018;Kuzmiak-Glancy et al, 2018). Rabbit oxymyoglobin (OxyMb), deoxymyoglobin (DeoxyMb) and metmyoglobin (MetMb) reference spectra were collected from commercially available rabbit heart myoglobin (Life Diagnostics, West Chester, PA, USA).…”

Section: Spectral Analysesmentioning

confidence: 96%

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Cardiac nitric oxide scavenging: role of myoglobin and mitochondria

Giles,

Edwards,

Covian

et al. 2023

The Journal of Physiology

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Vascular production of nitric oxide (NO) regulates vascular tone. However, highly permeable NO entering the cardiomyocyte would profoundly impact metabolism and signalling without scavenging mechanisms. The purpose of this study was to establish mechanisms of cardiac NO scavenging. Quantitative optical studies of normoxic working hearts demonstrated that micromolar NO concentrations did not alter mitochondria redox state or respiration despite detecting NO oxidation of oxymyoglobin to metmyoglobin. These data are consistent with proposals that the myoglobin/myoglobin reductase (Mb/MbR) system is the major NO scavenging site. However, kinetic studies in intact hearts reveal a minor role (∼9%) for the Mb/MbR system in NO scavenging. In vitro, oxygenated mitochondria studies confirm that micromolar concentrations of NO bind cytochrome oxidase (COX) and inhibit respiration. Mitochondria had a very high capacity for NO scavenging, importantly, independent of NO binding to COX. NO is also known to quickly react with reactive oxygen species (ROS) in vitro. Stimulation of NO scavenging with antimycin and its inhibition by substrate depletion are consistent with NO interacting with ROS generated in Complex I or III under aerobic conditions. Extrapolating these in vitro data to the intact heart supports the hypothesis that mitochondria are a major site of cardiac NO scavenging. imageKey points Cardiomyocyte scavenging of vascular nitric oxide (NO) is critical in maintaining normal cardiac function. Myoglobin redox cycling via myoglobin reductase has been proposed as a major NO scavenging site in the heart. Non‐invasive optical spectroscopy was used to monitor the effect of NO on mitochondria and myoglobin redox state in intact beating heart and isolated mitochondria. These non‐invasive studies reveal myoglobin/myoglobin reductase plays a minor role in cardiac NO scavenging. A high capacity for NO scavenging by heart mitochondria was demonstrated, independent of cytochrome oxidase binding but dependent on oxygen and high redox potentials consistent with generation of reactive oxygen species.

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Section: Resultsmentioning

confidence: 99%

Section: Spectral Analysesmentioning

confidence: 96%

Cardiac nitric oxide scavenging: role of myoglobin and mitochondria

Giles,

Edwards,

Covian

et al. 2023

The Journal of Physiology

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“…Furthermore, the mitoSOX signal increase in early reperfusion was inhibited by S1QEL, a specific inhibitor of ROS generation at Cx-I 59 , so we consider any contribution from other poorly-characterized oxidation sources to be minimal. Secondly, the mitoSOX signal is impacted both by its distribution between extracellular, cytosolic and mitochondrial compartments 37 , and by the primary and secondary filter effects from endogenous chromophores 47, 55 (see methods section 2.4). However, our use of fully-oxidized mitoSOX as a control (Supplementary Figure 1) ensures that any changes in the mitoSOX signal we observed originated only from in-situ probe oxidation, and not from redistribution of the probe or changes in the absorbance of myoglobin, cytochromes, etc.…”

Section: Discussionmentioning

confidence: 99%

“…Although similar spectrofluorometric cardiac perfusion apparatus has previously been constructed 32,[44][45][46][47][48] , prior efforts have used larger animal hearts (rats, guinea pigs) or have not simultaneously measured cardiac function. To the best of our knowledge, this is the first study of mouse hearts with simultaneous fluorescence and functional assessment.…”

Section: Cardiac Fluorescencementioning

confidence: 99%

Inhibiting succinate release worsens cardiac reperfusion injury by enhancing mitochondrial reactive oxygen species generation

Milliken¹,

Nadtochiy

Brookes

2022

Preprint

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The metabolite succinate accumulates during cardiac ischemia. Within 5 min. of reperfusion, succinate returns to baseline levels via both its release from cells and oxidation by mitochondrial complex II (Cx-II). The latter drives reactive oxygen species (ROS) generation and subsequent opening of the mitochondrial permeability transition (PT) pore, leading to cell death. Targeting succinate dynamics (accumulation/oxidation/release) may be therapeutically beneficial in cardiac ischemia-reperfusion (IR) injury. It has been proposed that blocking monocarboxylate transporter 1 (MCT-1) may be beneficial in IR, by preventing succinate release and subsequent engagement of downstream inflammatory signaling pathways. In contrast, herein we hypothesized that blocking MCT-1 would retain succinate in cells, exacerbating ROS generation and IR injury. Using the mitochondrial ROS probe mitoSOX, and a custom-built murine heart perfusion rig built into a spectrofluorometer, we measured ROS generation in-situ during the first moments of reperfusion, and found that acute MCT-1 inhibition enhanced mitochondrial ROS generation at reperfusion, and worsened IR injury (recovery of function and infarct size). Both these effects were abrogated by tandem inhibition of Cx-II, suggesting that succinate retention worsens IR due to driving more mitochondrial ROS generation. Furthermore, using the PT pore inhibitor cyclosporin A, along with monitoring of PT pore opening via the mitochondrial membrane potential indicator TMRE, we herein provide evidence that ROS generation during early reperfusion is upstream of the PT pore, not downstream as proposed by others. In addition, pore opening was exacerbated by MCT-1 inhibition. Together, these findings highlight the importance of succinate dynamics and mitochondrial ROS generation, as key determinants of PT pore opening and IR injury outcomes.

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“…Heart rate was calculated from the sinusoidal flow rate curve 36 The arrangement of the optics was previously detailed. 36,37 In brief, a Langendorff-perfused mouse heart was placed in a transparent glass chamber enclosed in a water jacket. The chamber had a white base to prevent direct illumination of the sensor.…”

Section: Langendorff Perfusion Of Mouse Heartsmentioning

confidence: 99%

MICU3 Regulates Mitochondrial Calcium and Cardiac Hypertrophy

Roman,

Mastoor,

Sun

et al. 2024

Circulation Research

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BACKGROUND: Calcium (Ca 2+ ) uptake by mitochondria occurs via the mitochondrial Ca 2+ uniporter. Mitochondrial Ca 2+ uniporter exists as a complex, regulated by 3 MICU (mitochondrial Ca 2+ uptake) proteins localized in the intermembrane space: MICU1, MICU2, and MICU3. Although MICU3 is present in the heart, its role is largely unknown. METHODS: We used CRISPR-Cas9 to generate a mouse with global deletion of MICU3 and an adeno-associated virus (AAV9) to overexpress MICU3 in wild-type mice. We examined the role of MICU3 in regulating mitochondrial calcium ([Ca 2+ ] m ) in ex vivo hearts using an optical method following adrenergic stimulation in perfused hearts loaded with a Ca 2+ -sensitive fluorophore. Additionally, we studied how deletion and overexpression of MICU3, respectively, impact cardiac function in vivo by echocardiography and the molecular composition of the mitochondrial Ca 2+ uniporter complex via Western blot, immunoprecipitation, and Blue native-PAGE analysis. Finally, we measured MICU3 expression in failing human hearts. RESULTS: Knock out MICU3 hearts and cardiomyocytes exhibited a significantly smaller increase in [Ca 2+ ] m than wild-type hearts following acute isoproterenol infusion. In contrast, overexpression of MICU3 hearts exhibited an enhanced increase in [Ca 2+ ] m compared with control hearts. Echocardiography analysis showed no significant difference in cardiac function in knock out MICU3 mice relative to wild-type mice at baseline. However, overexpression of MICU3 animals exhibited significantly reduced ejection fraction and fractional shortening compared with control mice. We observed a significant increase in the ratio of heart weight to tibia length in overexpression of MICU3 hearts compared with controls, consistent with hypertrophy. We also found a significant decrease in MICU3 protein and expression in failing human hearts. CONCLUSIONS: Our results indicate that increased and decreased expression of MICU3 enhances and reduces, respectively, the uptake of [Ca 2+ ] m in the heart. We conclude that MICU3 plays an important role in regulating [Ca 2+ ] m physiologically, and overexpression of MICU3 is sufficient to induce cardiac hypertrophy, making MICU3 a possible therapeutic target.

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Perfused murine heart optical transmission spectroscopy using optical catheter and integrating sphere: Effects of ischemia/reperfusion

Cited by 12 publications

References 36 publications

Cardiac nitric oxide scavenging: role of myoglobin and mitochondria

Cardiac nitric oxide scavenging: role of myoglobin and mitochondria

Inhibiting succinate release worsens cardiac reperfusion injury by enhancing mitochondrial reactive oxygen species generation

MICU3 Regulates Mitochondrial Calcium and Cardiac Hypertrophy

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