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Investigations into Mitochondrial Cell Death Mechanisms in Myocardial Infarction.

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Investigations into Mitochondrial Cell Death Mechanisms in Myocardial Infarction./
作者:	Axelrod, Joshua.
出版者:	Ann Arbor : ProQuest Dissertations & Theses, : 2023,
面頁冊數:	127 p.
附註:	Source: Dissertations Abstracts International, Volume: 85-06, Section: B.
Contained By:	Dissertations Abstracts International85-06B.
標題:	Cellular biology. -
電子資源:	https://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=30815157
ISBN:	9798381144581

Investigations into Mitochondrial Cell Death Mechanisms in Myocardial Infarction.
Axelrod, Joshua.

Investigations into Mitochondrial Cell Death Mechanisms in Myocardial Infarction. - Ann Arbor : ProQuest Dissertations & Theses, 2023 - 127 p.

Source: Dissertations Abstracts International, Volume: 85-06, Section: B.

Thesis (Ph.D.)--Albert Einstein College of Medicine, 2023.

Mitochondria are organelles found within cell that are involved in both metabolism and cell death. These effects on metabolism and cell death have an impact on development and disease pathogenesis. In this thesis I will present two distinct projects involving mitochondria and their roles in cell death during myocardial ischemia/reperfusion. The first project explores the impact of previously developed activators and inhibitors of mitofusins, proteins known to be involved in mitochondrial fusion and mitochondrial-ER proximity, in mice and determine their impact on cell death and infarct size in models of myocardial ischemia/reperfusion. In the second we will determine if the mitochondrial ATP synthase, the protein responsible for generation of most ATP in mammalian cells, also functions as the mitochondrial permeability transition pore, a hypothesized channel in the mitochondrial inner membrane that is thought to be involved in cell death. Mitochondria are dynamic organelles that undergo cycles of fission and fusion. These cycles of fission and fusion have been found to impact multiple cellular processes, including metabolism and cell death. Mitochondrial fusion is mediated by several GTPases including mitofusin 1 and 2 (MFN1 and MFN2), which are located on the outer mitochondrial membrane. MFN2, but not MFN1, has also been found on the endoplasmic/sarcoplasmic reticulum (ER/SR) surface and has been found to form tethering interactions with either MFN1 or MFN2 on the mitochondria to bring these organelles in proximity. This proximity between the ER and mitochondria modulates calcium ion transfer, mitophagy, ER stress, and phospholipid biosynthesis. Accumulation of mitochondrial Ca2+ can promote cardiomyocyte cell death during myocardial ischemia/reperfusion (reperfused myocardial infarction). Genetic deletion of either MFN1 or MFN2 reduces infarct size in myocardial ischemia/reperfusion. In previous work, pharmacological compounds (in the form of small molecules or cell permeable TAT-peptides) have been developed that affect mitofusin activity by manipulating their protein conformations. Among these compounds, small molecule MASM7 and peptide TAT-MAP, have been found to bind to MFN1 and MFN2, promoting their pro-fusion conformation and increasing their activity. Small molecules MFI-8 and peptide TAT-MIP bind to MFNs 1 and 2 to inhibit their mitochondrial fusion activity. The aim of this project in chapter 2 is to test whether modulation of endogenous mitofusins impacts cardiomyocyte death and, if so, to elucidate the underlying mechanisms and whether these agents could offer novel therapies for reducing infarct size in reperfused myocardial infarction.Abstract: Chapter 2Using mouse models of myocardial ischemia/reperfusion cardiomyocytes, we observed that activating MFNs with peptide TAT-MAP leads to an increase in infarct size, whereas inhibiting MFN peptide TAT-MIP has the opposite effect. Interestingly, the effects of these peptides were dependent on MFN2, but not MFN1, suggesting that they are mediated by effects on ER/SR-mitochondrial proximity. To assess the effects of MFN modulation on mitochondrial morphology and ER/SR-mitochondrial proximity, we utilized TEM imaging approaches in in-vivo heart tissues. Our results indicate that MFN activation augments ER/SR-mitochondrial proximity without significantly affecting mitochondrial morphology but TAT-MIP did not affect SR-mitochondrial proximity. Moreover, using FRET-based approaches in HEK-293 cells, we observed similar findings. Additionally, we found TAT-MAP promoted and TAT-MIP inhibited ER-mitochondria calcium transfer in histamine-stimulated MEFs in an MFN2-dependent manner. However, neither affected SR to mitochondrial calcium transfer in isolated adult cardiomyocytes. Further studies are underway to examine this in more depth. To evaluate the clinical potential of MFN inhibition in myocardial infarction, we also tested whether delayed administration of TAT-MIP following the period of ischemia (i.e., at the onset of reperfusion) would be effective in limiting cardiac damage. Our findings demonstrate similar reductions in infarct size with delayed dosing. The latter data suggest that pharmacological MFN inhibition may provide a therapy for reducing cardiac damage during reperfused myocardial infarction although the underlying mechanism remains unclear. Abstract: Chapter 3The mitochondrial permeability transition pore (mPTP) is a pore that has been experimentally observed on the mitochondrial intermembrane that is triggered by elevated concentrations of Ca2+ within the mitochondrial matrix. When triggered, mPTP has been found to cause mitochondrial swelling, the mobilization of apoptogens from the mitochondrial inner membrane and can cause cell death through apoptosis or necrotic mechanisms. Despite decades of research the molecular identity of the pore remains unknown. One of the candidates for mPTP has been mitochondrial ATP synthase, a complex of 28 proteins found on the mitochondrial inner membrane. The mitochondrial ATP synthase has been proposed by some to also function as the mPTP, while others have refuted this hypothesis using loss of function approaches to show that absence of the mitochondrial ATP synthase in cells has no effect on mPTP function. This, in turn, has led to the counter-hypothesis that redundant mPTP exists with the mitochondrial ATP synthase being one. In chapter 3, we present evidence showing that, rather than functioning as the mPTP, the mitochondrial ATP synthase is a negative regulator of this pore, thereby countering the notion that this protein complex functions as an mPTP.Consistent with previous work showing evidence against ATP synthase as the pore, we observed a persistence of mPTP in isolated mitochondria from and permeabilized HAP1 cells (nearly haploid leukemia cells) genetically engineered to lack assembled ATP synthase monomers or dimers. However, unlike previous findings, we found calcium-induced mPTP opening to be markedly sensitized in cells lacking assembled ATP synthase rather than this loss being a neutral event. Adding to this, we found that pharmacological or genetic deletion of cyclophilin D, a mitochondrial cis-trans prolyl isomerase known to promote mPTP opening desensitized this parameter to the same extent in cells lacking an assembled mitochondrial ATP synthase as in wild type cells. This observation suggests that cyclophilin D is acting in this function on substrates other than components of assembled ATP synthase. Furthermore, using patch clamping on mitoplasts, we found that mPTP channel conductance was unaffected by mitochondrial ATP synthase deletion and was still inhibited by cyclophilin D inhibition. Further, cardiac mitochondria isolated from mice that our lab engineered to be deficient in an assembled ATP synthase in cardiomyocytes also exhibited sensitization to calcium induced mPTP opening compared to wild type cardiac mitochondria and were desensitized to the same extent by cyclophilin D inhibition. Finally, mice with cardiomyocyte-specific ATP synthase depletion displayed larger infarcts when challenged with ischemia reperfusion in-vivo. In summary, we conclude that mitochondrial ATP synthase not function as mPTP, but rather serves as a negative regulator of this pore.

ISBN: 9798381144581Subjects--Topical Terms:

3172791
Cellular biology.
Subjects--Index Terms:

Cardiovascular

Investigations into Mitochondrial Cell Death Mechanisms in Myocardial Infarction.
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520 $a Mitochondria are organelles found within cell that are involved in both metabolism and cell death. These effects on metabolism and cell death have an impact on development and disease pathogenesis. In this thesis I will present two distinct projects involving mitochondria and their roles in cell death during myocardial ischemia/reperfusion. The first project explores the impact of previously developed activators and inhibitors of mitofusins, proteins known to be involved in mitochondrial fusion and mitochondrial-ER proximity, in mice and determine their impact on cell death and infarct size in models of myocardial ischemia/reperfusion. In the second we will determine if the mitochondrial ATP synthase, the protein responsible for generation of most ATP in mammalian cells, also functions as the mitochondrial permeability transition pore, a hypothesized channel in the mitochondrial inner membrane that is thought to be involved in cell death. Mitochondria are dynamic organelles that undergo cycles of fission and fusion. These cycles of fission and fusion have been found to impact multiple cellular processes, including metabolism and cell death. Mitochondrial fusion is mediated by several GTPases including mitofusin 1 and 2 (MFN1 and MFN2), which are located on the outer mitochondrial membrane. MFN2, but not MFN1, has also been found on the endoplasmic/sarcoplasmic reticulum (ER/SR) surface and has been found to form tethering interactions with either MFN1 or MFN2 on the mitochondria to bring these organelles in proximity. This proximity between the ER and mitochondria modulates calcium ion transfer, mitophagy, ER stress, and phospholipid biosynthesis. Accumulation of mitochondrial Ca2+ can promote cardiomyocyte cell death during myocardial ischemia/reperfusion (reperfused myocardial infarction). Genetic deletion of either MFN1 or MFN2 reduces infarct size in myocardial ischemia/reperfusion. In previous work, pharmacological compounds (in the form of small molecules or cell permeable TAT-peptides) have been developed that affect mitofusin activity by manipulating their protein conformations. Among these compounds, small molecule MASM7 and peptide TAT-MAP, have been found to bind to MFN1 and MFN2, promoting their pro-fusion conformation and increasing their activity. Small molecules MFI-8 and peptide TAT-MIP bind to MFNs 1 and 2 to inhibit their mitochondrial fusion activity. The aim of this project in chapter 2 is to test whether modulation of endogenous mitofusins impacts cardiomyocyte death and, if so, to elucidate the underlying mechanisms and whether these agents could offer novel therapies for reducing infarct size in reperfused myocardial infarction.Abstract: Chapter 2Using mouse models of myocardial ischemia/reperfusion cardiomyocytes, we observed that activating MFNs with peptide TAT-MAP leads to an increase in infarct size, whereas inhibiting MFN peptide TAT-MIP has the opposite effect. Interestingly, the effects of these peptides were dependent on MFN2, but not MFN1, suggesting that they are mediated by effects on ER/SR-mitochondrial proximity. To assess the effects of MFN modulation on mitochondrial morphology and ER/SR-mitochondrial proximity, we utilized TEM imaging approaches in in-vivo heart tissues. Our results indicate that MFN activation augments ER/SR-mitochondrial proximity without significantly affecting mitochondrial morphology but TAT-MIP did not affect SR-mitochondrial proximity. Moreover, using FRET-based approaches in HEK-293 cells, we observed similar findings. Additionally, we found TAT-MAP promoted and TAT-MIP inhibited ER-mitochondria calcium transfer in histamine-stimulated MEFs in an MFN2-dependent manner. However, neither affected SR to mitochondrial calcium transfer in isolated adult cardiomyocytes. Further studies are underway to examine this in more depth. To evaluate the clinical potential of MFN inhibition in myocardial infarction, we also tested whether delayed administration of TAT-MIP following the period of ischemia (i.e., at the onset of reperfusion) would be effective in limiting cardiac damage. Our findings demonstrate similar reductions in infarct size with delayed dosing. The latter data suggest that pharmacological MFN inhibition may provide a therapy for reducing cardiac damage during reperfused myocardial infarction although the underlying mechanism remains unclear. Abstract: Chapter 3The mitochondrial permeability transition pore (mPTP) is a pore that has been experimentally observed on the mitochondrial intermembrane that is triggered by elevated concentrations of Ca2+ within the mitochondrial matrix. When triggered, mPTP has been found to cause mitochondrial swelling, the mobilization of apoptogens from the mitochondrial inner membrane and can cause cell death through apoptosis or necrotic mechanisms. Despite decades of research the molecular identity of the pore remains unknown. One of the candidates for mPTP has been mitochondrial ATP synthase, a complex of 28 proteins found on the mitochondrial inner membrane. The mitochondrial ATP synthase has been proposed by some to also function as the mPTP, while others have refuted this hypothesis using loss of function approaches to show that absence of the mitochondrial ATP synthase in cells has no effect on mPTP function. This, in turn, has led to the counter-hypothesis that redundant mPTP exists with the mitochondrial ATP synthase being one. In chapter 3, we present evidence showing that, rather than functioning as the mPTP, the mitochondrial ATP synthase is a negative regulator of this pore, thereby countering the notion that this protein complex functions as an mPTP.Consistent with previous work showing evidence against ATP synthase as the pore, we observed a persistence of mPTP in isolated mitochondria from and permeabilized HAP1 cells (nearly haploid leukemia cells) genetically engineered to lack assembled ATP synthase monomers or dimers. However, unlike previous findings, we found calcium-induced mPTP opening to be markedly sensitized in cells lacking assembled ATP synthase rather than this loss being a neutral event. Adding to this, we found that pharmacological or genetic deletion of cyclophilin D, a mitochondrial cis-trans prolyl isomerase known to promote mPTP opening desensitized this parameter to the same extent in cells lacking an assembled mitochondrial ATP synthase as in wild type cells. This observation suggests that cyclophilin D is acting in this function on substrates other than components of assembled ATP synthase. Furthermore, using patch clamping on mitoplasts, we found that mPTP channel conductance was unaffected by mitochondrial ATP synthase deletion and was still inhibited by cyclophilin D inhibition. Further, cardiac mitochondria isolated from mice that our lab engineered to be deficient in an assembled ATP synthase in cardiomyocytes also exhibited sensitization to calcium induced mPTP opening compared to wild type cardiac mitochondria and were desensitized to the same extent by cyclophilin D inhibition. Finally, mice with cardiomyocyte-specific ATP synthase depletion displayed larger infarcts when challenged with ischemia reperfusion in-vivo. In summary, we conclude that mitochondrial ATP synthase not function as mPTP, but rather serves as a negative regulator of this pore.
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