Abstract P455: Acetylated Cyclophilin D Regulates Mitochondrial Electron Transport Chain Activity In The Developing Heart

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Gisela Beutner ◽  
Jonathan Burris ◽  
George A Porter
Keyword(s):  
Electron Transport ◽  
Enzymatic Activity ◽  
Electron Transport Chain ◽  
Isolated Heart ◽  
Permeability Transition ◽  
Mouse Heart ◽  
Cyclophilin D ◽  
Retention Capacity ◽  
Adult Heart ◽  
Transport Chain

Background: Mitochondria generate energy in form of ATP in mature eukaryotic cells. The components of the electron transport chain (ETC) are expressed at embryonic (E) day 9.5 in mouse hearts, but ETC activity and oxidative phosphorylation (OXPHOS) are uncoupled, because the permeability transition pore (PTP) is open. In mouse hearts the mPTP closes around E11.5. Opening and closing of the PTP is regulated by cyclophilin D (CypD), but the regulatory mechanism are not understood. Hypothesis: Maturation of ETC activity is regulated by acetylated CypD. Methods: Cardiac tissue homogenates or isolated heart mitochondria from mice, ranging in age from E 9.5 to adult, were used to measure oxygen consumption and the calcium retention capacity. The expression of proteins of the ETC and their assembly into supercomplexes was followed by denaturing and native electrophoresis. The expression of CypD and its acetylation status were assessed by western blotting and densitometry. The enzymatic activity of the ETC complexes and CypD was measured using spectrophotometry. Results: In the heart of mouse embryos at E 9.5 mitochondrial ETC activity and OXPHOS are not coupled (respiratory control ratio (RCR) 1.48 ± 0.17, n=9). Addition of 1 μM cyclosporin A, an inhibitor of CypD and the PTP acutely increases the RCR to 3.69 ± 0.59 (n=5). At E13.5, the end of the embryonic period in the mouse, OXPHOS is coupled and not significantly different from the adult heart. The enzymatic activity of the ETC complexes I, II, III and V increases significantly from E9.5 to the adult heart and the assembly of mitochondrial supercomplexes begins at about E13.5. The total expression of CypD increases as the mouse heart matures, but the enzymatic activity of CypD decreases. In addition, the ratio of acetylated CypD to the total expressed CypD decreased from 1.1 ± 0.16 (n=3) at E9.5 to 0.54 ± 0.06 (n=3) in the adult heart. Conclusion: The activity of acetylated CypD regulates maturation of mitochondrial ETC activity in the developing mouse heart.

Abstract P446: Cyclophilin D Inhibition Rescues Cardiac Function And Electron Transport Chain Assembly And Activity In Hypoxia Exposed Neonatal Mice

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Jonathan Burris ◽  
Gisela Beutner ◽  
George A Porter
Keyword(s):  
Electron Transport ◽  
Cardiac Function ◽  
Enzymatic Activity ◽  
Complex I ◽  
Electron Transport Chain ◽  
Critical Role ◽  
Heart Weight ◽  
Cyclophilin D ◽  
Neonatal Heart ◽  
Transport Chain

Introduction: Mitochondria play a critical role in cardiac myocyte physiology and differentiation. Hypoxia decreases cardiac function. Changes in embryonic heart metabolism at the level of the electron transport chain (ETC) are regulated by a chaperone protein known as cyclophilin D (CypD). Inhibition of CypD with chemicals such as cyclosporin A (CsA) and N-methyl-4-isoleucine cyclosporin (NIM811) leads to more complex mitochondrial structure and effective oxidative phosphorylation. Hypothesis: Inhibition of CypD with CsA or NIM811 will rescue the detrimental effects of hypoxia on cardiac function and on ETC assembly and activity. Methods: Mice were exposed to continuous hypoxia (12% oxygen) immediately before birth (gestational age E19.5) to postnatal day 7 (P7). Hypoxic mice received no treatment (No Tx) or intraperitoneal injections 10mg/kg of vehicle (VEH), CsA, or NIM811 from P1 to P6. Litters of mice born into room air served as controls. On P7, mice were anesthetized and underwent echocardiography and/or hearts were harvested for mitochondrial isolation. Enzymatic activity of ETC complexes was quantified using spectrophotometry and normalized to total protein. To measure physical assembly of complex I & V of the ETC, high resolution clear native polyacrylamide gel electrophoresis (HCRN PAGE) followed by in-gel assays were utilized using appropriate protein loading controls. Results: Cardiac ejection fraction was decreased in hypoxic No Tx (P<0.0001) and VEH (P<0.0001), but was rescued by CsA or NIM811 when compared to room air controls (P>0.05). Heart weight to body weight ratio was increased in No Tx and VEH groups (P< 0.0001) and rescued in the CsA and NIM811 groups when compared to room air controls (P>0.05). Complex I enzymatic activity was rescued with treatment with CsA and NIM811. HCRN PAGE followed by in-gel ETC complex assay demonstrated assembly of complexes I and V into dimers and tetramer in the room air, CsA, and NIM811 groups that was not seen in the No Tx and VEH groups. Conclusion: Pharmacologic inhibition of CypD reversed the effects of hypoxia on cardiac function and ETC activity and assembly in the neonatal heart. Our studies may help develop therapies to treat neonatal cardiomyopathies and the effects of hypoxia on the neonatal heart.

Membrane permeability transition as induced by dysfunction of the electron transport chain

IUBMB Life ◽  
1997 ◽  
Vol 41 (5) ◽  
pp. 961-968
Author(s):  
Edmundo Chávez ◽  
Esperanza Meléndez ◽  
Cecilia Zazueta ◽  
Horacio Reyes-Vivas ◽  
Sofía Perales
Keyword(s):  
Electron Transport ◽  
Membrane Permeability ◽  
Electron Transport Chain ◽  
Permeability Transition ◽  
Membrane Permeability Transition ◽  
Transport Chain

Abstract 331: Impact of Acetylated Cyclophilin D on the Mitochondrial Permeability Transition Pore

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Gisela Beutner ◽  
Jacob Perkins ◽  
Ronak A Sardari ◽  
George A Porter
Keyword(s):  
Atp Synthase ◽  
Cyclosporine A ◽  
Matrix Protein ◽  
Mitochondrial Permeability Transition ◽  
Atp Synthesis ◽  
Permeability Transition Pore ◽  
Permeability Transition ◽  
Cyclophilin D ◽  
Retention Capacity ◽  
Transport Chain

Background: The mitochondrial matrix protein cyclophilin D (CypD) is a key regulator of mitochondrial function. CypD controls electron transport chain activity and ATP synthesis by regulating the permeability transition pore (PTP). The activity of CypD is regulated by several post-translational modifications including acetylation of lysine 166 in the mouse. Objective: To investigate how acetylation at lysine 166 of CypD specifically in the heart modifies its ability to regulate the PTP and the ATP synthase. Results: We generated a conditional cardiac knock-in mouse model where lysine 166 has been mutated into glutamine (CypD K166Q ) to mimic permanent acetylation of CypD. The mice were either +/+, +/- or -/- for the expression of native CypD. Results show that mitochondrial oxygen consumption was not affected by the expression of CypD K166Q . The calcium retention capacity (CRC) was measured with Arsenazo III and decreased significantly when CypD K166Q was expressed. The CypD inhibitor cyclosporine A significantly increased the CRC in WT mice. However, cyclosporine A was did not inhibit CypD in the hearts of mice expressing only CypD K166Q or in addition to wild-type CypD. The ability of the ATP synthase to create dimers or oligomers was assessed by western blotting and the hydrolysis of ATP in in-gel assays and shows that expression of CypD K166Q decreased the assembly of the ATP synthase into dimers or oligomers. Conclusions: Our data show that the expression of CypD K166Q increases the sensitivity of PTP opening to calcium and limits the assembly of ATP synthase into oligomers.

scholarly journals Cyclophilin D, Somehow a Master Regulator of Mitochondrial Function

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 176 ◽  
Author(s):  
George A. Porter ◽  
Gisela Beutner
Keyword(s):  
Electron Transport ◽  
Mitochondrial Function ◽  
Electron Transport Chain ◽  
Mitochondrial Permeability Transition ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Mitochondrial Protein ◽  
Cyclophilin D ◽  
Master Regulator ◽  
Transport Chain

Cyclophilin D (CyPD) is an important mitochondrial chaperone protein whose mechanism of action remains a mystery. It is well known for regulating mitochondrial function and coupling of the electron transport chain and ATP synthesis by controlling the mitochondrial permeability transition pore (PTP), but more recent evidence suggests that it may regulate electron transport chain activity. Given its identification as a peptidyl-prolyl, cis-trans isomerase (PPIase), CyPD, is thought to be involved in mitochondrial protein folding, but very few reports demonstrate the presence of this activity. By contrast, CyPD may also perform a scaffolding function, as it binds to a number of important proteins in the mitochondrial matrix and inner mitochondrial membrane. From a clinical perspective, inhibiting CyPD to inhibit PTP opening protects against ischemia–reperfusion injury, making modulation of CyPD activity a potentially important therapeutic goal, but the lack of knowledge about the mechanisms of CyPD’s actions remains problematic for such therapies. Thus, the important yet enigmatic nature of CyPD somehow makes it a master regulator, yet a troublemaker, for mitochondrial function.

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