BackgroundFor a long time, mitochondrial electron transport chain (ETC) complexes was thought to ... more BackgroundFor a long time, mitochondrial electron transport chain (ETC) complexes was thought to function as separate entities. However, studies conducted in the past decade revealed that individual ETC complexes can assemble to form supramolecular structures known as supercomplexes (SCs). The SCs, particularly respirasome are composed of the ETC complexes I, III and IV in various stoichiometry. The respirasome is considered to play an important role in facilitating electron transport, reducing ROS production, and maintaining structural integrity of individual ETC complexes. Despite extensive studies on the molecular identity, the physiological role of SCs in cells, particularly, in high energy‐consuming organs such as the heart and brain remain unknown. These studies seek to determine whether disassembly of SCs affects the cardiac function in rats.MethodsHearts isolated from Sprague Dawley rats (275–325g) were perfused with Krebs Henseleit solution (KHS) via the Langendorff‐perfusi...
Optic atrophy type 1 protein (OPA1), a dynamin-related GTPase, that, in addition to mitochondrial... more Optic atrophy type 1 protein (OPA1), a dynamin-related GTPase, that, in addition to mitochondrial fusion, plays an important role in maintaining the structural organization and integrity of the inner mitochondrial membrane (IMM). OPA1 exists in two forms: IMM-bound long-OPA1 (L-OPA1) and soluble short-OPA1 (S-OPA1), a product of L-OPA1 proteolytic cleavage localized in the intermembrane space. In addition to OPA1, the structural and functional integrity of IMM can be regulated by changes in the matrix volume due to the opening/closure of permeability transition pores (PTP). Herein, we investigated the crosstalk between the PTP and OPA1 to clarify whether PTP opening is involved in OPA1-mediated regulation of respiratory chain supercomplexes (RCS) assembly using cardiac mitochondria and cell line. We found that: 1) Proteolytic cleavage of L-OPA1 is stimulated by PTP-induced mitochondrial swelling, 2) OPA1 knockdown reduces PTP-induced mitochondrial swelling but enhances ROS production, 3) OPA1 deficiency impairs the RCS assembly associated with diminished ETC activity and oxidative phosphorylation, 4) OPA1 has no physical interaction with phospholipid scramblase 3 although OPA1 downregulation increases expression of the scramblase. Thus, this study demonstrates that L-OPA1 cleavage depends on the PTP-induced mitochondrial swelling suggesting a regulatory role of the PTP-OPA1 axis in RCS assembly and mitochondrial bioenergetics.
Introduction: Leptin is a 16 kDa protein produced primarily by adipose tissue but recently we dem... more Introduction: Leptin is a 16 kDa protein produced primarily by adipose tissue but recently we demonstrated that it is also synthesized in heart and cardiomyocytes. In addition, we identified leptin...
Iron is an important ion that is mainly involved in different aspects of cellular metabolism. Nor... more Iron is an important ion that is mainly involved in different aspects of cellular metabolism. Normally, iron homeostasis is regulated by storage and transport proteins that help stabilize and facilitate transport throughout the body. Transferrin is the main iron transport protein in the blood; it transports ferric iron (Fe3+) from absorption sites to different tissue cells via endocytosis upon interacting with the transferrin receptor. In the cell, in addition to the cytoplasm, mitochondria are heavily involved in iron metabolism. The excess of labile (free unbound) iron in the cell can undergo the Fenton reaction to further enhance ROS production inevitably leading to cell death through apoptosis and/or ferroptosis. Ferroptosis is a new type of cell death that is mediated by iron-dependent lipid peroxidation and is associated with a myriad of diseases such as cardiovascular and neurodegenerative diseases, and cancer, among others. Mitochondria are involved in ferroptotic cell death signaling, nevertheless, the exact mechanisms underlying the contribution of mitochondria to ferroptotic signaling remain unknown. This study was performed to elucidate the effects of iron on cell viability and mitochondrial function in cardiomyocytes. H9c2 cardiomyocytes were cultured in the presence/absence of ferric ammonium citrate (FAC, 200 and 400 μM), tert-butyl hydroxide (TBH, 10 and 100 μM), and deferoxamine (DFO, 200 and 400 μM) individually and in combination. Cell death was quantified by the lactate dehydrogenase (LDH) activity in the culture media whereas mitochondrial function was estimated by changes in the mitochondrial membrane potential measured by using TMRM, a potential-sensitive fluorescent dye. Our results show that TBH at low concentrations or FAC at both low and high concentrations did not induce cell death. However, FAC at high concentration (400 μM) in combination with TBH (10 μM) significantly increased cell death. Additionally, DFO alone induced cell death, demonstrating the importance of maintaining iron balance in cellular media. In conclusion, this study demonstrated that the detrimental effects of iron (FAC) on cell viability are increased in the presence of oxidative stress (TBH) in H9c2 cardiomyocytes. This study was supported by the National Institutes of Health (Grant R16GM145390). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
BackgroundMitochondria are the bioenergetic engines of the cell, thus maintaining mitochondrial h... more BackgroundMitochondria are the bioenergetic engines of the cell, thus maintaining mitochondrial health is essential for meeting energy demands. Mitochondrial function is achieved upon the balance of structural processes that involve mitochondrial biogenesis, fusion and fission. Structural and functional integrity of the mitochondrial membranes, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM) is important for the maintenance of the mitochondrial quality control and mitochondrial metabolism. Therefore, elucidation of the individual contribution of each membrane to mitochondrial metabolism and function, as well as mitochondria‐mediated cell survival pathways and cell death is important. In this study, we attempted to set up the methods for isolation of mitoplasts (mitochondria without OMM) and the OMM from the rat heart.MethodsCardiac mitochondria were isolated from Sprague Dawley rats (250–300g) by differential centrifugation and purified via Percoll gradient (continuous layers of 19%, 30% and 60% concentration). Isolation of mitoplasts was performed by incubation in different hypotonic and hypertonic buffers, sonication (20% output/power), and separation of membranes by ultracentrifugation using sucrose continuous gradients (20–80%). Mitochondrial swelling assay was made to confirm the swelling/shrinking method used. Mitoplast formation was validated by monoamine oxidase (MAO) activity, oxygen consumption in the presence and absence of cytochrome‐c and Western Blotting against specific inner and outer mitochondrial membrane proteins.ResultsWestern Blot analysis showed pure isolated mitochondria, validated by the absence of GAPDH (cytosolic marker) upon Percoll gradient. In addition, more than 30% decrease in TOMM20 (OMM specific protein) expression in isolated mitoplasts in comparison to mitochondria. Mitochondrial respiratory rates and MAO activity suggests changes in mitochondrial membrane distribution. Mitochondrial swelling assay showed that cardiac mitochondria were resistant to swelling in hypotonic solution, therefore other methods involving the addition of Ca+2 or EGTA were used to induced separation of mitochondrial membranes.ConclusionDespite application of different preparative approaches, we were not able to isolate purified OMM, and only one method was proved to make a slight reduction of OMM contamination in mitoplasts.Support or Funding InformationSupported by the NIGMS NIH grant SC1GM128210This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondria, known as the powerhouse of the cell, also participate in the regulation of ion home... more Mitochondria, known as the powerhouse of the cell, also participate in the regulation of ion homeostasis, redox status, cell growth and differentiation, lipid metabolism, and cell death. The inner mitochondrial membrane (IMM) plays a crucial role in the regulation of mitochondrial metabolism and function. The dynamin-related GTPase optic atrophy-1 (OPA1) localized in the IMM is responsible for mitochondria fusion. In addition, OPA1 has been shown to play an important role in maintaining the structural integrity and functional activity of mitochondrial cristae. We have recently shown that mitochondrial swelling stimulates enzymatic cleavage of OPA1 by OMA1, a zinc metallopeptidase, which converts long-OPA1 (L-OPA1) to short-OPA1 (S-OPA1). Cleavage of L-OPA1 was suggested to diminish mitochondrial fusion and bioenergetics leading to cell death. The role of OPA1 cleavage and OMA1 activation under diverse swelling conditions has not yet been elucidated. In this study, we investigated the role of OPA1 cleavage under various mitochondrial swelling conditions in the presence and absence of Myls22 (an OPA1 inhibitor), and TPEN (a zinc-chelator and OMA1 inhibitor). Cardiac mitochondria were isolated from Sprague Dawley rats and their function was assessed under Ca2+-induced mitochondrial swelling with oxoglutarate and malate (OM) with/without ADP. Our dose-dependent experiments revealed that Myls22 and TPEN exert the maximum effect at 50 and 5 μM, respectively. Next, we found that the maximum respiration rate (state 3) of mitochondria for complex I is diminished by both inhibitors. However, neither Myls22 nor TPEN prevented alterations in mitochondrial respiration rates, swelling, membrane potential, and calcium retention capacity induced by Ca2+. Finally, we evaluated the protein expression of L-OPA1 via western blot and found more OPA1 cleavage under state 3 compared to state 2 respiration rate suggesting the regulatory role of S-OPA1 in mitochondrial respiration. Thus, our data demonstrate that although mitochondrial swelling stimulates L-OPA cleavage associated with mitochondrial dysfunction, inhibition of OPA1 cleavage/OMA1 activity by Mysl22/TPEN is ineffective in ameliorating the effects of Ca2+-induced mitochondria swelling. This study was supported by the National Science Foundation (Grant 2006477). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
The assembly of mitochondrial electron transport chain (ETC) supercomplexes (SCs), particularly t... more The assembly of mitochondrial electron transport chain (ETC) supercomplexes (SCs), particularly the respirasome containing complexes I, III, and IV have been shown to participate in facilitating electron transport, reducing ROS production and maintaining the structural integrity of individual ETC complexes. However, the physiological role of SCs in high energy demanding tissues such as the heart remains unknown. Here, we elucidated whether disassembly of SCs affects the cardiac function. Hearts isolated from adult male Sprague Dawley rats were perfused using a Langendorff-mode perfusion with Krebs-Henseleit solution (KHS) for the first 20 min (equilibration period) followed by perfusion in the following groups: (i) 40-min perfusion with KHS (n=4), (ii, iii) 20-min perfusion with rotenone (an inducer of SC dissociation) or vehicle (ethanol, n=8 for both), and (iv, v) 20-min perfusion with rotenone or vehicle followed by a 20-min perfusion with KHS without rotenone or vehicle (n=6 for both). Cardiac function was monitored throughout the entire perfusion period. At the end of each protocol, the mitochondria were isolated for analysis of SCs, permeability transition, respiration rates, and ROS production. We found that cardiac function between rats perfused with KHS (i) and ethanol (i, iii) had no significant difference, though rotenone perfused rats (ii and iv) had a significant reduction (<40%) in cardiac function associated with reduced oxygen consumption rates in these groups. Furthermore, a significant increase in ROS production (ii) and permeability transition pore opening (iv) was observed when the hearts were perfused with rotenone with and without subsequent washout, respectively. Analysis of SCs by blue native PAGE displayed a significant reduction in SC levels in both ethanol (iii and v) and rotenone (ii and iv) perfused rats. Surprisingly, there were no differences in SC levels between the ethanol (iii and v) and rotenone (ii and iv) perfused groups. In addition, incubation of isolated intact mitochondria with ethanol and acetaldehyde did not demonstrate any direct effects of these compounds on ETC SC assembly. In conclusion, our data demonstrate a lack of an associative link between mitochondrial ETC SCs and cardiac function in rats.
Optic atrophy-1 (OPA1) plays a crucial role in the regulation of mitochondria fusion and particip... more Optic atrophy-1 (OPA1) plays a crucial role in the regulation of mitochondria fusion and participates in maintaining the structural integrity of mitochondrial cristae. Here we elucidate the role of OPA1 cleavage induced by calcium swelling in the presence of Myls22 (an OPA1 GTPase activity inhibitor) and TPEN (an OMA1 inhibitor). The rate of ADP-stimulated respiration was found diminished by both inhibitors, and they did not prevent Ca2+-induced mitochondrial respiratory dysfunction, membrane depolarization, or swelling. L-OPA1 cleavage was stimulated at state 3 respiration; therefore, our data suggest that L-OPA1 cleavage produces S-OPA1 to maintain mitochondrial bioenergetics in response to stress.
Mitochondrion, known as the “powerhouse” of the cell, regulates ion homeostasis, redox state, cel... more Mitochondrion, known as the “powerhouse” of the cell, regulates ion homeostasis, redox state, cell proliferation and differentiation, and lipid synthesis. The inner mitochondrial membrane (IMM) controls mitochondrial metabolism and function. It possesses high levels of proteins that account for ~70% of the membrane mass and are involved in the electron transport chain, oxidative phosphorylation, energy transfer, and ion transport, among others. The mitochondrial matrix volume plays a crucial role in IMM remodeling. Several ion transport mechanisms, particularly K+ and Ca2+, regulate matrix volume. Small increases in matrix volume through IMM alterations can activate mitochondrial respiration, whereas excessive swelling can impair the IMM topology and initiates mitochondria-mediated cell death. The opening of mitochondrial permeability transition pores, the well-characterized phenomenon with unknown molecular identity, in low- and high-conductance modes are involved in physiological ...
Background: An increasing number of experimental and clinical studies show a link between Alzheim... more Background: An increasing number of experimental and clinical studies show a link between Alzheimer’s disease and heart diseases such as heart failure, ischemic heart disease, and atrial fibrillation. However, the mechanisms underlying the potential role of amyloid-β (Aβ) in the pathogenesis of cardiac dysfunction in Alzheimer’s disease remain unknown. We have recently shown the effects of Aβ1 - 40 and Aβ1 - 42 on cell viability and mitochondrial function in cardiomyocytes and coronary artery endothelial cells. Objective: In this study, we investigated the effects of Aβ1 - 40 and Aβ1 - 42 on the metabolism of cardiomyocytes and coronary artery endothelial cells. Methods: Gas chromatography-mass spectrometry was used to analyze metabolomic profiles of cardiomyocytes and coronary artery endothelial cells treated with Aβ1 - 40 and Aβ1 - 42. In addition, we determined mitochondrial respiration and lipid peroxidation in these cells. Results: We found that the metabolism of different amin...
BackgroundFor a long time, mitochondrial electron transport chain (ETC) complexes was thought to ... more BackgroundFor a long time, mitochondrial electron transport chain (ETC) complexes was thought to function as separate entities. However, studies conducted in the past decade revealed that individual ETC complexes can assemble to form supramolecular structures known as supercomplexes (SCs). The SCs, particularly respirasome are composed of the ETC complexes I, III and IV in various stoichiometry. The respirasome is considered to play an important role in facilitating electron transport, reducing ROS production, and maintaining structural integrity of individual ETC complexes. Despite extensive studies on the molecular identity, the physiological role of SCs in cells, particularly, in high energy‐consuming organs such as the heart and brain remain unknown. These studies seek to determine whether disassembly of SCs affects the cardiac function in rats.MethodsHearts isolated from Sprague Dawley rats (275–325g) were perfused with Krebs Henseleit solution (KHS) via the Langendorff‐perfusi...
Optic atrophy type 1 protein (OPA1), a dynamin-related GTPase, that, in addition to mitochondrial... more Optic atrophy type 1 protein (OPA1), a dynamin-related GTPase, that, in addition to mitochondrial fusion, plays an important role in maintaining the structural organization and integrity of the inner mitochondrial membrane (IMM). OPA1 exists in two forms: IMM-bound long-OPA1 (L-OPA1) and soluble short-OPA1 (S-OPA1), a product of L-OPA1 proteolytic cleavage localized in the intermembrane space. In addition to OPA1, the structural and functional integrity of IMM can be regulated by changes in the matrix volume due to the opening/closure of permeability transition pores (PTP). Herein, we investigated the crosstalk between the PTP and OPA1 to clarify whether PTP opening is involved in OPA1-mediated regulation of respiratory chain supercomplexes (RCS) assembly using cardiac mitochondria and cell line. We found that: 1) Proteolytic cleavage of L-OPA1 is stimulated by PTP-induced mitochondrial swelling, 2) OPA1 knockdown reduces PTP-induced mitochondrial swelling but enhances ROS production, 3) OPA1 deficiency impairs the RCS assembly associated with diminished ETC activity and oxidative phosphorylation, 4) OPA1 has no physical interaction with phospholipid scramblase 3 although OPA1 downregulation increases expression of the scramblase. Thus, this study demonstrates that L-OPA1 cleavage depends on the PTP-induced mitochondrial swelling suggesting a regulatory role of the PTP-OPA1 axis in RCS assembly and mitochondrial bioenergetics.
Introduction: Leptin is a 16 kDa protein produced primarily by adipose tissue but recently we dem... more Introduction: Leptin is a 16 kDa protein produced primarily by adipose tissue but recently we demonstrated that it is also synthesized in heart and cardiomyocytes. In addition, we identified leptin...
Iron is an important ion that is mainly involved in different aspects of cellular metabolism. Nor... more Iron is an important ion that is mainly involved in different aspects of cellular metabolism. Normally, iron homeostasis is regulated by storage and transport proteins that help stabilize and facilitate transport throughout the body. Transferrin is the main iron transport protein in the blood; it transports ferric iron (Fe3+) from absorption sites to different tissue cells via endocytosis upon interacting with the transferrin receptor. In the cell, in addition to the cytoplasm, mitochondria are heavily involved in iron metabolism. The excess of labile (free unbound) iron in the cell can undergo the Fenton reaction to further enhance ROS production inevitably leading to cell death through apoptosis and/or ferroptosis. Ferroptosis is a new type of cell death that is mediated by iron-dependent lipid peroxidation and is associated with a myriad of diseases such as cardiovascular and neurodegenerative diseases, and cancer, among others. Mitochondria are involved in ferroptotic cell death signaling, nevertheless, the exact mechanisms underlying the contribution of mitochondria to ferroptotic signaling remain unknown. This study was performed to elucidate the effects of iron on cell viability and mitochondrial function in cardiomyocytes. H9c2 cardiomyocytes were cultured in the presence/absence of ferric ammonium citrate (FAC, 200 and 400 μM), tert-butyl hydroxide (TBH, 10 and 100 μM), and deferoxamine (DFO, 200 and 400 μM) individually and in combination. Cell death was quantified by the lactate dehydrogenase (LDH) activity in the culture media whereas mitochondrial function was estimated by changes in the mitochondrial membrane potential measured by using TMRM, a potential-sensitive fluorescent dye. Our results show that TBH at low concentrations or FAC at both low and high concentrations did not induce cell death. However, FAC at high concentration (400 μM) in combination with TBH (10 μM) significantly increased cell death. Additionally, DFO alone induced cell death, demonstrating the importance of maintaining iron balance in cellular media. In conclusion, this study demonstrated that the detrimental effects of iron (FAC) on cell viability are increased in the presence of oxidative stress (TBH) in H9c2 cardiomyocytes. This study was supported by the National Institutes of Health (Grant R16GM145390). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
BackgroundMitochondria are the bioenergetic engines of the cell, thus maintaining mitochondrial h... more BackgroundMitochondria are the bioenergetic engines of the cell, thus maintaining mitochondrial health is essential for meeting energy demands. Mitochondrial function is achieved upon the balance of structural processes that involve mitochondrial biogenesis, fusion and fission. Structural and functional integrity of the mitochondrial membranes, the outer mitochondrial membrane (OMM) and the inner mitochondrial membrane (IMM) is important for the maintenance of the mitochondrial quality control and mitochondrial metabolism. Therefore, elucidation of the individual contribution of each membrane to mitochondrial metabolism and function, as well as mitochondria‐mediated cell survival pathways and cell death is important. In this study, we attempted to set up the methods for isolation of mitoplasts (mitochondria without OMM) and the OMM from the rat heart.MethodsCardiac mitochondria were isolated from Sprague Dawley rats (250–300g) by differential centrifugation and purified via Percoll gradient (continuous layers of 19%, 30% and 60% concentration). Isolation of mitoplasts was performed by incubation in different hypotonic and hypertonic buffers, sonication (20% output/power), and separation of membranes by ultracentrifugation using sucrose continuous gradients (20–80%). Mitochondrial swelling assay was made to confirm the swelling/shrinking method used. Mitoplast formation was validated by monoamine oxidase (MAO) activity, oxygen consumption in the presence and absence of cytochrome‐c and Western Blotting against specific inner and outer mitochondrial membrane proteins.ResultsWestern Blot analysis showed pure isolated mitochondria, validated by the absence of GAPDH (cytosolic marker) upon Percoll gradient. In addition, more than 30% decrease in TOMM20 (OMM specific protein) expression in isolated mitoplasts in comparison to mitochondria. Mitochondrial respiratory rates and MAO activity suggests changes in mitochondrial membrane distribution. Mitochondrial swelling assay showed that cardiac mitochondria were resistant to swelling in hypotonic solution, therefore other methods involving the addition of Ca+2 or EGTA were used to induced separation of mitochondrial membranes.ConclusionDespite application of different preparative approaches, we were not able to isolate purified OMM, and only one method was proved to make a slight reduction of OMM contamination in mitoplasts.Support or Funding InformationSupported by the NIGMS NIH grant SC1GM128210This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Mitochondria, known as the powerhouse of the cell, also participate in the regulation of ion home... more Mitochondria, known as the powerhouse of the cell, also participate in the regulation of ion homeostasis, redox status, cell growth and differentiation, lipid metabolism, and cell death. The inner mitochondrial membrane (IMM) plays a crucial role in the regulation of mitochondrial metabolism and function. The dynamin-related GTPase optic atrophy-1 (OPA1) localized in the IMM is responsible for mitochondria fusion. In addition, OPA1 has been shown to play an important role in maintaining the structural integrity and functional activity of mitochondrial cristae. We have recently shown that mitochondrial swelling stimulates enzymatic cleavage of OPA1 by OMA1, a zinc metallopeptidase, which converts long-OPA1 (L-OPA1) to short-OPA1 (S-OPA1). Cleavage of L-OPA1 was suggested to diminish mitochondrial fusion and bioenergetics leading to cell death. The role of OPA1 cleavage and OMA1 activation under diverse swelling conditions has not yet been elucidated. In this study, we investigated the role of OPA1 cleavage under various mitochondrial swelling conditions in the presence and absence of Myls22 (an OPA1 inhibitor), and TPEN (a zinc-chelator and OMA1 inhibitor). Cardiac mitochondria were isolated from Sprague Dawley rats and their function was assessed under Ca2+-induced mitochondrial swelling with oxoglutarate and malate (OM) with/without ADP. Our dose-dependent experiments revealed that Myls22 and TPEN exert the maximum effect at 50 and 5 μM, respectively. Next, we found that the maximum respiration rate (state 3) of mitochondria for complex I is diminished by both inhibitors. However, neither Myls22 nor TPEN prevented alterations in mitochondrial respiration rates, swelling, membrane potential, and calcium retention capacity induced by Ca2+. Finally, we evaluated the protein expression of L-OPA1 via western blot and found more OPA1 cleavage under state 3 compared to state 2 respiration rate suggesting the regulatory role of S-OPA1 in mitochondrial respiration. Thus, our data demonstrate that although mitochondrial swelling stimulates L-OPA cleavage associated with mitochondrial dysfunction, inhibition of OPA1 cleavage/OMA1 activity by Mysl22/TPEN is ineffective in ameliorating the effects of Ca2+-induced mitochondria swelling. This study was supported by the National Science Foundation (Grant 2006477). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
The assembly of mitochondrial electron transport chain (ETC) supercomplexes (SCs), particularly t... more The assembly of mitochondrial electron transport chain (ETC) supercomplexes (SCs), particularly the respirasome containing complexes I, III, and IV have been shown to participate in facilitating electron transport, reducing ROS production and maintaining the structural integrity of individual ETC complexes. However, the physiological role of SCs in high energy demanding tissues such as the heart remains unknown. Here, we elucidated whether disassembly of SCs affects the cardiac function. Hearts isolated from adult male Sprague Dawley rats were perfused using a Langendorff-mode perfusion with Krebs-Henseleit solution (KHS) for the first 20 min (equilibration period) followed by perfusion in the following groups: (i) 40-min perfusion with KHS (n=4), (ii, iii) 20-min perfusion with rotenone (an inducer of SC dissociation) or vehicle (ethanol, n=8 for both), and (iv, v) 20-min perfusion with rotenone or vehicle followed by a 20-min perfusion with KHS without rotenone or vehicle (n=6 for both). Cardiac function was monitored throughout the entire perfusion period. At the end of each protocol, the mitochondria were isolated for analysis of SCs, permeability transition, respiration rates, and ROS production. We found that cardiac function between rats perfused with KHS (i) and ethanol (i, iii) had no significant difference, though rotenone perfused rats (ii and iv) had a significant reduction (<40%) in cardiac function associated with reduced oxygen consumption rates in these groups. Furthermore, a significant increase in ROS production (ii) and permeability transition pore opening (iv) was observed when the hearts were perfused with rotenone with and without subsequent washout, respectively. Analysis of SCs by blue native PAGE displayed a significant reduction in SC levels in both ethanol (iii and v) and rotenone (ii and iv) perfused rats. Surprisingly, there were no differences in SC levels between the ethanol (iii and v) and rotenone (ii and iv) perfused groups. In addition, incubation of isolated intact mitochondria with ethanol and acetaldehyde did not demonstrate any direct effects of these compounds on ETC SC assembly. In conclusion, our data demonstrate a lack of an associative link between mitochondrial ETC SCs and cardiac function in rats.
Optic atrophy-1 (OPA1) plays a crucial role in the regulation of mitochondria fusion and particip... more Optic atrophy-1 (OPA1) plays a crucial role in the regulation of mitochondria fusion and participates in maintaining the structural integrity of mitochondrial cristae. Here we elucidate the role of OPA1 cleavage induced by calcium swelling in the presence of Myls22 (an OPA1 GTPase activity inhibitor) and TPEN (an OMA1 inhibitor). The rate of ADP-stimulated respiration was found diminished by both inhibitors, and they did not prevent Ca2+-induced mitochondrial respiratory dysfunction, membrane depolarization, or swelling. L-OPA1 cleavage was stimulated at state 3 respiration; therefore, our data suggest that L-OPA1 cleavage produces S-OPA1 to maintain mitochondrial bioenergetics in response to stress.
Mitochondrion, known as the “powerhouse” of the cell, regulates ion homeostasis, redox state, cel... more Mitochondrion, known as the “powerhouse” of the cell, regulates ion homeostasis, redox state, cell proliferation and differentiation, and lipid synthesis. The inner mitochondrial membrane (IMM) controls mitochondrial metabolism and function. It possesses high levels of proteins that account for ~70% of the membrane mass and are involved in the electron transport chain, oxidative phosphorylation, energy transfer, and ion transport, among others. The mitochondrial matrix volume plays a crucial role in IMM remodeling. Several ion transport mechanisms, particularly K+ and Ca2+, regulate matrix volume. Small increases in matrix volume through IMM alterations can activate mitochondrial respiration, whereas excessive swelling can impair the IMM topology and initiates mitochondria-mediated cell death. The opening of mitochondrial permeability transition pores, the well-characterized phenomenon with unknown molecular identity, in low- and high-conductance modes are involved in physiological ...
Background: An increasing number of experimental and clinical studies show a link between Alzheim... more Background: An increasing number of experimental and clinical studies show a link between Alzheimer’s disease and heart diseases such as heart failure, ischemic heart disease, and atrial fibrillation. However, the mechanisms underlying the potential role of amyloid-β (Aβ) in the pathogenesis of cardiac dysfunction in Alzheimer’s disease remain unknown. We have recently shown the effects of Aβ1 - 40 and Aβ1 - 42 on cell viability and mitochondrial function in cardiomyocytes and coronary artery endothelial cells. Objective: In this study, we investigated the effects of Aβ1 - 40 and Aβ1 - 42 on the metabolism of cardiomyocytes and coronary artery endothelial cells. Methods: Gas chromatography-mass spectrometry was used to analyze metabolomic profiles of cardiomyocytes and coronary artery endothelial cells treated with Aβ1 - 40 and Aβ1 - 42. In addition, we determined mitochondrial respiration and lipid peroxidation in these cells. Results: We found that the metabolism of different amin...
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