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Aging-dependent changes in rat heart mitochondrial glutaredoxins--Implications for redox regulation.

Gao XH, Qanungo S, Pai HV, Starke DW, Steller KM, Fujioka H, Lesnefsky EJ, Kerner J, Rosca MG, Hoppel CL, Mieyal JJ - Redox Biol (2013)

Bottom Line: Recently we found that aging-dependent increase in susceptibility of cardiomyocytes to apoptosis was attributable to decrease in cytosolic glutaredoxin 1 (Grx1) and concomitant decrease in NF-κB-mediated expression of anti-apoptotic proteins.We found that the inactive Grx2 can be fully reactivated by sodium dithionite or exogenous superoxide production mediated by xanthine oxidase.However, treatment with rotenone, which generates intramitochondrial superoxide through inhibition of mitochondrial respiratory chain Complex I, did not lead to Grx2 activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Division of Cardiology, Case Western Reserve University, School of Medicine, Cleveland, OH 44106, USA.

ABSTRACT
Clinical and animal studies have documented that hearts of the elderly are more susceptible to ischemia/reperfusion damage compared to young adults. Recently we found that aging-dependent increase in susceptibility of cardiomyocytes to apoptosis was attributable to decrease in cytosolic glutaredoxin 1 (Grx1) and concomitant decrease in NF-κB-mediated expression of anti-apoptotic proteins. Besides primary localization in the cytosol, Grx1 also exists in the mitochondrial intermembrane space (IMS). In contrast, Grx2 is confined to the mitochondrial matrix. Here we report that Grx1 is decreased by 50-60% in the IMS, but Grx2 is increased by 1.4-2.6 fold in the matrix of heart mitochondria from elderly rats. Determination of in situ activities of the Grx isozymes from both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria revealed that Grx1 was fully active in the IMS. However, Grx2 was mostly in an inactive form in the matrix, consistent with reversible sequestration of the active-site cysteines of two Grx2 molecules in complex with an iron-sulfur cluster. Our quantitative evaluations of the active/inactive ratio for Grx2 suggest that levels of dimeric Grx2 complex with iron-sulfur clusters are increased in SSM and IFM in the hearts of elderly rats. We found that the inactive Grx2 can be fully reactivated by sodium dithionite or exogenous superoxide production mediated by xanthine oxidase. However, treatment with rotenone, which generates intramitochondrial superoxide through inhibition of mitochondrial respiratory chain Complex I, did not lead to Grx2 activation. These findings suggest that insufficient ROS accumulates in the vicinity of dimeric Grx2 to activate it in situ.

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Mitochondrial Grx1 content is decreased in both populations of heart mitochondria from elderly Fischer 344 rats. Mitochondrial Grx1 contents in the SSM and IFM from young adult and elderly F344 rat heart tissues were determined by interpolation of the band density of t-Bid-released Grx1, using a standard curve relating the densities of the bands of several different amounts of purified human Grx1 protein that were analyzed on the same gels in each respective case, as described previously [12]. The values are reported as ng Grx1 per mg of mitochondrial protein (mean±SEM, n=5) for (A) SSM and (B) IFM. Diminutions of Grx1 content were observed in the IFM and SSM from the elderly rats compared to the young adult rats. The diminution of mitochondrial Grx1 content in the elderly F344 rat hearts was corroborated using gold labeling of Grx1 antibodies based on electron microscopy analysis (C) and (D). Black arrows indicate Grx1 localization in the outer mitochondrial compartment (intermembrane space) of heart mitochondria from (C) young adult and (D) elderly F344 rats. Twenty-two gold particles were counted in the mitochondrial sample from young adult rats, compared to ten that were observed in the sample from the elderly rats.
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f0010: Mitochondrial Grx1 content is decreased in both populations of heart mitochondria from elderly Fischer 344 rats. Mitochondrial Grx1 contents in the SSM and IFM from young adult and elderly F344 rat heart tissues were determined by interpolation of the band density of t-Bid-released Grx1, using a standard curve relating the densities of the bands of several different amounts of purified human Grx1 protein that were analyzed on the same gels in each respective case, as described previously [12]. The values are reported as ng Grx1 per mg of mitochondrial protein (mean±SEM, n=5) for (A) SSM and (B) IFM. Diminutions of Grx1 content were observed in the IFM and SSM from the elderly rats compared to the young adult rats. The diminution of mitochondrial Grx1 content in the elderly F344 rat hearts was corroborated using gold labeling of Grx1 antibodies based on electron microscopy analysis (C) and (D). Black arrows indicate Grx1 localization in the outer mitochondrial compartment (intermembrane space) of heart mitochondria from (C) young adult and (D) elderly F344 rats. Twenty-two gold particles were counted in the mitochondrial sample from young adult rats, compared to ten that were observed in the sample from the elderly rats.

Mentions: We found an age-associated decrease in Grx1 content in both the SSM (~50%) and the IFM (~60%) (Fig. 2A and B). Corroborating the diminution of Grx1 in the IMS in situ, we used immunocytochemistry with gold particles and electron microscopy to localize and quantify the Grx1 in isolated heart mitochondria (Fig. 2C and D). Thus, fewer gold particle-conjugated Grx1 antibodies were observed in the outer compartment and contiguous intracristal space (IMS) of the mitochondria isolated from an elderly rat as compared to young adult. The extent of diminution of Grx1 in the IMS is comparable to the diminution of Grx1 that we observed previously in the cytosol [18], suggesting that the content of Grx1 undergoes dynamic equilibration between the cytosol and IMS during aging (see Discussion section). In contrast, the content of Grx2 was increased in the SSM (1.4-fold) and more substantially in the IFM (2.6-fold) from elderly rats as compared to young adult rats (Fig. 3A and B), suggesting different regulatory mechanisms governing the contents of mitochondrial Grx1 and Grx2. We next investigated whether the changes in Grx1 and Grx2 content corresponded to changes in their catalytic deglutathionylase activities.


Aging-dependent changes in rat heart mitochondrial glutaredoxins--Implications for redox regulation.

Gao XH, Qanungo S, Pai HV, Starke DW, Steller KM, Fujioka H, Lesnefsky EJ, Kerner J, Rosca MG, Hoppel CL, Mieyal JJ - Redox Biol (2013)

Mitochondrial Grx1 content is decreased in both populations of heart mitochondria from elderly Fischer 344 rats. Mitochondrial Grx1 contents in the SSM and IFM from young adult and elderly F344 rat heart tissues were determined by interpolation of the band density of t-Bid-released Grx1, using a standard curve relating the densities of the bands of several different amounts of purified human Grx1 protein that were analyzed on the same gels in each respective case, as described previously [12]. The values are reported as ng Grx1 per mg of mitochondrial protein (mean±SEM, n=5) for (A) SSM and (B) IFM. Diminutions of Grx1 content were observed in the IFM and SSM from the elderly rats compared to the young adult rats. The diminution of mitochondrial Grx1 content in the elderly F344 rat hearts was corroborated using gold labeling of Grx1 antibodies based on electron microscopy analysis (C) and (D). Black arrows indicate Grx1 localization in the outer mitochondrial compartment (intermembrane space) of heart mitochondria from (C) young adult and (D) elderly F344 rats. Twenty-two gold particles were counted in the mitochondrial sample from young adult rats, compared to ten that were observed in the sample from the elderly rats.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4127417&req=5

f0010: Mitochondrial Grx1 content is decreased in both populations of heart mitochondria from elderly Fischer 344 rats. Mitochondrial Grx1 contents in the SSM and IFM from young adult and elderly F344 rat heart tissues were determined by interpolation of the band density of t-Bid-released Grx1, using a standard curve relating the densities of the bands of several different amounts of purified human Grx1 protein that were analyzed on the same gels in each respective case, as described previously [12]. The values are reported as ng Grx1 per mg of mitochondrial protein (mean±SEM, n=5) for (A) SSM and (B) IFM. Diminutions of Grx1 content were observed in the IFM and SSM from the elderly rats compared to the young adult rats. The diminution of mitochondrial Grx1 content in the elderly F344 rat hearts was corroborated using gold labeling of Grx1 antibodies based on electron microscopy analysis (C) and (D). Black arrows indicate Grx1 localization in the outer mitochondrial compartment (intermembrane space) of heart mitochondria from (C) young adult and (D) elderly F344 rats. Twenty-two gold particles were counted in the mitochondrial sample from young adult rats, compared to ten that were observed in the sample from the elderly rats.
Mentions: We found an age-associated decrease in Grx1 content in both the SSM (~50%) and the IFM (~60%) (Fig. 2A and B). Corroborating the diminution of Grx1 in the IMS in situ, we used immunocytochemistry with gold particles and electron microscopy to localize and quantify the Grx1 in isolated heart mitochondria (Fig. 2C and D). Thus, fewer gold particle-conjugated Grx1 antibodies were observed in the outer compartment and contiguous intracristal space (IMS) of the mitochondria isolated from an elderly rat as compared to young adult. The extent of diminution of Grx1 in the IMS is comparable to the diminution of Grx1 that we observed previously in the cytosol [18], suggesting that the content of Grx1 undergoes dynamic equilibration between the cytosol and IMS during aging (see Discussion section). In contrast, the content of Grx2 was increased in the SSM (1.4-fold) and more substantially in the IFM (2.6-fold) from elderly rats as compared to young adult rats (Fig. 3A and B), suggesting different regulatory mechanisms governing the contents of mitochondrial Grx1 and Grx2. We next investigated whether the changes in Grx1 and Grx2 content corresponded to changes in their catalytic deglutathionylase activities.

Bottom Line: Recently we found that aging-dependent increase in susceptibility of cardiomyocytes to apoptosis was attributable to decrease in cytosolic glutaredoxin 1 (Grx1) and concomitant decrease in NF-κB-mediated expression of anti-apoptotic proteins.We found that the inactive Grx2 can be fully reactivated by sodium dithionite or exogenous superoxide production mediated by xanthine oxidase.However, treatment with rotenone, which generates intramitochondrial superoxide through inhibition of mitochondrial respiratory chain Complex I, did not lead to Grx2 activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Division of Cardiology, Case Western Reserve University, School of Medicine, Cleveland, OH 44106, USA.

ABSTRACT
Clinical and animal studies have documented that hearts of the elderly are more susceptible to ischemia/reperfusion damage compared to young adults. Recently we found that aging-dependent increase in susceptibility of cardiomyocytes to apoptosis was attributable to decrease in cytosolic glutaredoxin 1 (Grx1) and concomitant decrease in NF-κB-mediated expression of anti-apoptotic proteins. Besides primary localization in the cytosol, Grx1 also exists in the mitochondrial intermembrane space (IMS). In contrast, Grx2 is confined to the mitochondrial matrix. Here we report that Grx1 is decreased by 50-60% in the IMS, but Grx2 is increased by 1.4-2.6 fold in the matrix of heart mitochondria from elderly rats. Determination of in situ activities of the Grx isozymes from both subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria revealed that Grx1 was fully active in the IMS. However, Grx2 was mostly in an inactive form in the matrix, consistent with reversible sequestration of the active-site cysteines of two Grx2 molecules in complex with an iron-sulfur cluster. Our quantitative evaluations of the active/inactive ratio for Grx2 suggest that levels of dimeric Grx2 complex with iron-sulfur clusters are increased in SSM and IFM in the hearts of elderly rats. We found that the inactive Grx2 can be fully reactivated by sodium dithionite or exogenous superoxide production mediated by xanthine oxidase. However, treatment with rotenone, which generates intramitochondrial superoxide through inhibition of mitochondrial respiratory chain Complex I, did not lead to Grx2 activation. These findings suggest that insufficient ROS accumulates in the vicinity of dimeric Grx2 to activate it in situ.

Show MeSH
Related in: MedlinePlus