Optimization of measurement of mitochondrial electron transport activity in postmortem human brain samples and measurement of susceptibility to rotenone and 4-hydroxynonenal inhibition

Benavides, Gloria A.; Mueller, Toni; Darley‐Usmar, Victor; Zhang, Jianhua

doi:10.1016/j.redox.2022.102241

Cited by 7 publications

(4 citation statements)

References 48 publications

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“…CS and lactate dehydrogenase assays were measured spectrophotometrically as previously described. 34 , 35 For CS activity, 20 mmol/L oxaloacetate, 10 mmol/L acetyl‐coenzyme A (CoA), and 5,5‐dithiobis (2,4‐nitrobenzoic acid) were added with the heart homogenate (10 μg) in 0.1% Triton X‐100 and 100 mmol/L Tris (pH 8.0) buffer, then measured the product of 20 mmol/L 5,5‐dithiobis (2,4‐nitrobenzoic acid)–‐CoA at 412 nm for kinetic absorbance change for nmol/min per mg protein at 37 °C. For lactate dehydrogenase activities, 0.3 mmol/L NADH and 10 mmol/L pyruvate were added in heart homogenate (10 μg) in PBS (pH 7.4) buffer, then measured NADH oxidation kinetics at 340 nm and calculated the activity as nmol/min per mg protein at 37 °C.…”

Section: Methodsmentioning

confidence: 99%

Sustained Increases in Cardiomyocyte Protein O ‐Linked β‐N‐Acetylglucosamine Levels Lead to Cardiac Hypertrophy and Reduced Mitochondrial Function Without Systolic Contractile Impairment

Ha,

Bakshi,

Brahma

et al. 2023

JAHA

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Background Lifestyle and metabolic diseases influence the severity and pathogenesis of cardiovascular disease through numerous mechanisms, including regulation via posttranslational modifications. A specific posttranslational modification, the addition of O ‐linked β‐ N acetylglucosamine ( O ‐GlcNAcylation), has been implicated in molecular mechanisms of both physiological and pathologic adaptations. The current study aimed to test the hypothesis that in cardiomyocytes, sustained protein O ‐GlcNAcylation contributes to cardiac adaptations, and its progression to pathophysiology. Methods and Results Using a naturally occurring dominant‐negative O ‐GlcNAcase (dnOGA) inducible cardiomyocyte‐specific overexpression transgenic mouse model, we induced dnOGA in 8‐ to 10‐week‐old mouse hearts. We examined the effects of 2‐week and 24‐week dnOGA overexpression, which progressed to a 1.8‐fold increase in protein O‐ GlcNAcylation. Two‐week increases in protein O ‐GlcNAc levels did not alter heart weight or function; however, 24‐week increases in protein O ‐GlcNAcylation led to cardiac hypertrophy, mitochondrial dysfunction, fibrosis, and diastolic dysfunction. Interestingly, systolic function was maintained in 24‐week dnOGA overexpression, despite several changes in gene expression associated with cardiovascular disease. Specifically, mRNA‐sequencing analysis revealed several gene signatures, including reduction of mitochondrial oxidative phosphorylation, fatty acid, and glucose metabolism pathways, and antioxidant response pathways after 24‐week dnOGA overexpression. Conclusions This study indicates that moderate increases in cardiomyocyte protein O ‐GlcNAcylation leads to a differential response with an initial reduction of metabolic pathways (2‐week), which leads to cardiac remodeling (24‐week). Moreover, the mouse model showed evidence of diastolic dysfunction consistent with a heart failure with preserved ejection fraction. These findings provide insight into the adaptive versus maladaptive responses to increased O‐ GlcNAcylation in heart.

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Section: Methodsmentioning

confidence: 99%

Sustained Increases in Cardiomyocyte Protein O ‐Linked β‐N‐Acetylglucosamine Levels Lead to Cardiac Hypertrophy and Reduced Mitochondrial Function Without Systolic Contractile Impairment

Ha,

Bakshi,

Brahma

et al. 2023

JAHA

Self Cite

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“…To each well, cytochrome c and alamethicin were added as described (Acin‐Perez et al, 2020; Benavides et al, 2022). To measure complex activities, substrates were added: NADH for complex I, succinate and rotenone for complex II, duroquinol for complex III, and ascorbate with TMPD (N,N,N′,N′‐tetramethyl‐para‐phenylene‐diamine) for complex IV as described (Acin‐Perez et al, 2020; Benavides et al, 2022). Activities were terminated by respective complex inhibitors including rotenone for complex I, antimycin A for complexes II and III, and azide for complex IV as described (Acin‐Perez et al, 2020; Benavides et al, 2022).…”

Section: Methodsmentioning

confidence: 99%

“…The reaction product DTNB‐CoA was monitored at 412 nm and the decrease of NADH was measured with absorption at 340 nm. The changes of absorbance were used to calculate activities as nmol/min/mg protein (Acin‐Perez et al, 2020; Benavides et al, 2022; Van et al, 2022).…”

Section: Methodsmentioning

confidence: 99%

“…The plate was then centrifuged at 2000 g for 20 min at 4°C. To each well, cytochrome c and alamethicin were added as described (Acin‐Perez et al, 2020; Benavides et al, 2022). To measure complex activities, substrates were added: NADH for complex I, succinate and rotenone for complex II, duroquinol for complex III, and ascorbate with TMPD (N,N,N′,N′‐tetramethyl‐para‐phenylene‐diamine) for complex IV as described (Acin‐Perez et al, 2020; Benavides et al, 2022).…”

Section: Methodsmentioning

confidence: 99%

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