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Sarco-endoplasmic reticulum ATPase participates in the regulation of mitochondrial calcium
A. Newman / Free Radical Biology and Medicine 128 (2018) S21–S46
dietary intake potentiates RSV infection and severe disease with associated mitochondrial metabolic disruption and oxidative stress.
https://doi.org/10.1016/j.freeradbiomed.2018.10.019
18
Sarco-endoplasmic reticulum ATPase participates in the regulation of mitochondrial calcium Jena Goodmann, Dominique Croteau, Michael Kirber, Deborah Siwik, Ivan Luptak, David Pimentel, Wilson Colucci Boston University, USA
Background: Although sarco-endoplasmic reticulum ATPase-2 (SERCA) is located in close proximity to mitochondria, its role in the regulation of mitochondrial calcium ([Ca2 þ ]m) is not clear. Reactive oxygen species (ROS) such as H2O2 can oxidize SERCA at C674, thereby inhibiting its activity and potentially affecting [Ca2 þ ]m in conditions associated with oxidative stress in the myocardium. Methods: In adult rat ventricular myocytes (ARVM) we used the SERCA inhibitor thapsigargin (TG) or H2O2 to inhibit SERCA and measured the effects on [Ca2 þ ]m using a mitochondrially-targeted [Ca2 þ ] probe (CAMmito). CAMmito was excited at 446nm and emission was detected at 480nm/535nm to derive a CFP/YFP ratio. WT and C674S SERCA, and CAMmito were expressed in (ARVM) using adenoviral vectors. Results: TG (1 uM, 30 min) increased the CFP/YFP ratio 11 þ5% (p ¼0.07, n¼ 4). H2O2 (100 uM, 10 min) similarly increased the ratio by 11 þ1% (p ¼0.02, n ¼7). The basal CFP/YFP ratio was not different in ARVM expressing WT vs. C674S SERCA (p ¼ 0.89, n¼4). In ARVM expressing WT SERCA, H2O2 increased the CFP/YFP ratio 21 þ4% (p ¼ 0.02, n¼4), whereas in ARVM expressing C674S SERCA the H2O2-stimulated increase was decreased to 12 þ%, (p¼ 0.03 vs. baseline, 0.2 vs. WT SERCA, n¼4). Conclusion: In cardiac myocytes, SERCA inhibition with TG increased [Ca2 þ ]m, indicating that SERCA participates in regulation of [Ca2 þ ]m under basal conditions. ROS also increased [Ca2 þ ]m, although to a greater magnitude (21 vs. 11%) than TG. The redox-insensitive SERCA mutant C674S decreased the ROS-stimulated increase to 12%, suggesting that oxidative inhibition of SERCA contributes to the ROS-stimulated increase in [Ca2 þ ]m. SERCA may be an important regulator of [Ca2 þ ]m in cardiac myocytes in health and in disease states associated with increased oxidative stress.
https://doi.org/10.1016/j.freeradbiomed.2018.10.020
19
Apocynin reduces pressor effects of Ang II in SHR
and
vasoconstrictor
Murilo Graton 1, n, Simone Regina Potje 2, Jéssica Antonini Troiano 1, Gabriel Tavares Vale 2, Priscila Scarpim Benevides 1, Carlos Renato Tirapelli 2, Ana Cláudia Nakamune 1, Lusiane Maria Bendhack 2, Cristina Antoniali 1 1 2
São Paulo State University (UNESP), Brazil University of São Paulo, Brazil
S27
NAD(P)H oxidase (NOX) activity and expression can be activated by angiotensin (Ang) II. We have previously observed that apocynin, a NOX inhibitor, prevented endotelial dysfunction and reduced blood pressure by increasing nitric oxide (NO) and reducing ROS concentrations in endothelial cells. We evaluated the effect of apocynin on the contractile responses to Ang II in resistance arteries of SHR and the mechanisms involved on these effects. SHR were treated from the 4th to the 10th week of life with apocynin (30 mg/Kg). Wistar rats were used as normotensive control. Using mesenteric arteries, we performed concentration-response curves to Ang II and determined eNOS and NOX isoforms and subunits expressions, lucigenin chemiluminescence and nitrate/nitrite levels. Data were expressed as mean 7 SEM. Apocynin increased endothelium modulation and/or NOS activity on Ang II vasoconstrictor responses in mesenteric arteries of SHR. Treatment with apocynin did not alter NO synthase activity, and eNOS, NOX1, NOXO1, and NOX4 expressions, however, it decreased NOX2 and p47phox expressions in mesenteric beds of SHR treated. Moreover, treatment apocynin was able to decrease ROS production in these vessels. The lower reactivity of Ang II in resistance arteries would lead to lower peripheral vascular resistance and consequently the reduction of mean arterial pressure and Ang II pressor effect in SHR treated with apocynin, as previously observed. All these results demonstrated that in vivo treatment with apocynin induces important alterations of several mechanisms that lead to the reduction of the pressor and vasoconstrictor effects of Ang II in SHR. Apocynin effect involves further mechanisms besides the modulation of vascular ROS, which improve NO availability in vascular cells of SHR. Ethics Comittee CEUA FOA 450/2015. Financial Support FAPESP 2016/22180-9, CAPES and CNPq.
https://doi.org/10.1016/j.freeradbiomed.2018.10.021
20
PKM2 S-glutathionylation promotes the reprogramming of macrophage metabolism and activation states Joshua Hayesn, Luxi Wang, Yong Joo Ahn, Reto Asmis Wake Forest School of Medicine, USA
Introduction: Pyruvate Kinase Isoform M2 (PKM2) regulates glycolysis in monocytes and macrophages and is induced and S-glutathionylated in response to diet-induced metabolic stress. We hypothesized that PKM2 S-glutathionylation promotes PKM2 tetramer dissociation into glycolytically-inactive dimers and that nuclear translocation of these dimers activates HIF1a-dependent gene expression, inducing both glycolytic enzymes as well and pro-inflammatory proteins, reprogramming macrophages to a hyper-inflammatory phenotype. Methods: Bone marrow derived macrophages (BMDM) overexpressing glutaredoxin-1-EGFP (Grx1) or EGFP only were exposed for 36 h to metabolic stress (high glucose plus native human LDL). PKM2 expression and phosphorylation state (Tyr105) were assessed in cell lysates by Western blot analysis. PKM2 S-glutathionylation was quantified using the Biotinswitch assay. PKM2 oligomerization state was measured by non-reducing SDS-PAGE and Western blot analysis. To confirm the role of PKM2 S-glutathionylation on metabolic reprogramming of monocytes in vivo, we conducted lentiviral gene transfer experiments in mice using CD68 promoter-driven Grx1 and EGFP expression. Mice were fed a HFD for 10 week to induce monocyte priming. We purified blood monocytes from EGFPMactg and Grx1Mactg mice and determined by single-cell Western blot PKM2 induction and phosphorylation, GLUT1, 14-3-3z and IL-1b expression.
dietary intake potentiates RSV infection and severe disease with associated mitochondrial metabolic disruption and oxidative stress.
https://doi.org/10.1016/j.freeradbiomed.2018.10.019
18
Sarco-endoplasmic reticulum ATPase participates in the regulation of mitochondrial calcium Jena Goodmann, Dominique Croteau, Michael Kirber, Deborah Siwik, Ivan Luptak, David Pimentel, Wilson Colucci Boston University, USA
Background: Although sarco-endoplasmic reticulum ATPase-2 (SERCA) is located in close proximity to mitochondria, its role in the regulation of mitochondrial calcium ([Ca2 þ ]m) is not clear. Reactive oxygen species (ROS) such as H2O2 can oxidize SERCA at C674, thereby inhibiting its activity and potentially affecting [Ca2 þ ]m in conditions associated with oxidative stress in the myocardium. Methods: In adult rat ventricular myocytes (ARVM) we used the SERCA inhibitor thapsigargin (TG) or H2O2 to inhibit SERCA and measured the effects on [Ca2 þ ]m using a mitochondrially-targeted [Ca2 þ ] probe (CAMmito). CAMmito was excited at 446nm and emission was detected at 480nm/535nm to derive a CFP/YFP ratio. WT and C674S SERCA, and CAMmito were expressed in (ARVM) using adenoviral vectors. Results: TG (1 uM, 30 min) increased the CFP/YFP ratio 11 þ5% (p ¼0.07, n¼ 4). H2O2 (100 uM, 10 min) similarly increased the ratio by 11 þ1% (p ¼0.02, n ¼7). The basal CFP/YFP ratio was not different in ARVM expressing WT vs. C674S SERCA (p ¼ 0.89, n¼4). In ARVM expressing WT SERCA, H2O2 increased the CFP/YFP ratio 21 þ4% (p ¼ 0.02, n¼4), whereas in ARVM expressing C674S SERCA the H2O2-stimulated increase was decreased to 12 þ%, (p¼ 0.03 vs. baseline, 0.2 vs. WT SERCA, n¼4). Conclusion: In cardiac myocytes, SERCA inhibition with TG increased [Ca2 þ ]m, indicating that SERCA participates in regulation of [Ca2 þ ]m under basal conditions. ROS also increased [Ca2 þ ]m, although to a greater magnitude (21 vs. 11%) than TG. The redox-insensitive SERCA mutant C674S decreased the ROS-stimulated increase to 12%, suggesting that oxidative inhibition of SERCA contributes to the ROS-stimulated increase in [Ca2 þ ]m. SERCA may be an important regulator of [Ca2 þ ]m in cardiac myocytes in health and in disease states associated with increased oxidative stress.
https://doi.org/10.1016/j.freeradbiomed.2018.10.020
19
Apocynin reduces pressor effects of Ang II in SHR
and
vasoconstrictor
Murilo Graton 1, n, Simone Regina Potje 2, Jéssica Antonini Troiano 1, Gabriel Tavares Vale 2, Priscila Scarpim Benevides 1, Carlos Renato Tirapelli 2, Ana Cláudia Nakamune 1, Lusiane Maria Bendhack 2, Cristina Antoniali 1 1 2
São Paulo State University (UNESP), Brazil University of São Paulo, Brazil
S27
NAD(P)H oxidase (NOX) activity and expression can be activated by angiotensin (Ang) II. We have previously observed that apocynin, a NOX inhibitor, prevented endotelial dysfunction and reduced blood pressure by increasing nitric oxide (NO) and reducing ROS concentrations in endothelial cells. We evaluated the effect of apocynin on the contractile responses to Ang II in resistance arteries of SHR and the mechanisms involved on these effects. SHR were treated from the 4th to the 10th week of life with apocynin (30 mg/Kg). Wistar rats were used as normotensive control. Using mesenteric arteries, we performed concentration-response curves to Ang II and determined eNOS and NOX isoforms and subunits expressions, lucigenin chemiluminescence and nitrate/nitrite levels. Data were expressed as mean 7 SEM. Apocynin increased endothelium modulation and/or NOS activity on Ang II vasoconstrictor responses in mesenteric arteries of SHR. Treatment with apocynin did not alter NO synthase activity, and eNOS, NOX1, NOXO1, and NOX4 expressions, however, it decreased NOX2 and p47phox expressions in mesenteric beds of SHR treated. Moreover, treatment apocynin was able to decrease ROS production in these vessels. The lower reactivity of Ang II in resistance arteries would lead to lower peripheral vascular resistance and consequently the reduction of mean arterial pressure and Ang II pressor effect in SHR treated with apocynin, as previously observed. All these results demonstrated that in vivo treatment with apocynin induces important alterations of several mechanisms that lead to the reduction of the pressor and vasoconstrictor effects of Ang II in SHR. Apocynin effect involves further mechanisms besides the modulation of vascular ROS, which improve NO availability in vascular cells of SHR. Ethics Comittee CEUA FOA 450/2015. Financial Support FAPESP 2016/22180-9, CAPES and CNPq.
https://doi.org/10.1016/j.freeradbiomed.2018.10.021
20
PKM2 S-glutathionylation promotes the reprogramming of macrophage metabolism and activation states Joshua Hayesn, Luxi Wang, Yong Joo Ahn, Reto Asmis Wake Forest School of Medicine, USA
Introduction: Pyruvate Kinase Isoform M2 (PKM2) regulates glycolysis in monocytes and macrophages and is induced and S-glutathionylated in response to diet-induced metabolic stress. We hypothesized that PKM2 S-glutathionylation promotes PKM2 tetramer dissociation into glycolytically-inactive dimers and that nuclear translocation of these dimers activates HIF1a-dependent gene expression, inducing both glycolytic enzymes as well and pro-inflammatory proteins, reprogramming macrophages to a hyper-inflammatory phenotype. Methods: Bone marrow derived macrophages (BMDM) overexpressing glutaredoxin-1-EGFP (Grx1) or EGFP only were exposed for 36 h to metabolic stress (high glucose plus native human LDL). PKM2 expression and phosphorylation state (Tyr105) were assessed in cell lysates by Western blot analysis. PKM2 S-glutathionylation was quantified using the Biotinswitch assay. PKM2 oligomerization state was measured by non-reducing SDS-PAGE and Western blot analysis. To confirm the role of PKM2 S-glutathionylation on metabolic reprogramming of monocytes in vivo, we conducted lentiviral gene transfer experiments in mice using CD68 promoter-driven Grx1 and EGFP expression. Mice were fed a HFD for 10 week to induce monocyte priming. We purified blood monocytes from EGFPMactg and Grx1Mactg mice and determined by single-cell Western blot PKM2 induction and phosphorylation, GLUT1, 14-3-3z and IL-1b expression.