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Hepatology (Baltimore, Md.) Jan 2023Increased megamitochondria formation and impaired mitophagy in hepatocytes have been linked to the pathogenesis of alcohol-associated liver disease (ALD). This study...
BACKGROUND AND AIMS
Increased megamitochondria formation and impaired mitophagy in hepatocytes have been linked to the pathogenesis of alcohol-associated liver disease (ALD). This study aims to determine the mechanisms by which alcohol consumption increases megamitochondria formation in the pathogenesis of ALD.
APPROACH AND RESULTS
Human alcoholic hepatitis (AH) liver samples were used for electron microscopy, histology, and biochemical analysis. Liver-specific dynamin-related protein 1 (DRP1; gene name DNM1L, an essential gene regulating mitochondria fission ) knockout (L-DRP1 KO) mice and wild-type mice were subjected to chronic plus binge alcohol feeding. Both human AH and alcohol-fed mice had decreased hepatic DRP1 with increased accumulation of hepatic megamitochondria. Mechanistic studies revealed that alcohol feeding decreased DRP1 by impairing transcription factor EB-mediated induction of DNM1L . L-DRP1 KO mice had increased megamitochondria and decreased mitophagy with increased liver injury and inflammation, which were further exacerbated by alcohol feeding. Seahorse flux and unbiased metabolomics analysis showed alcohol intake increased mitochondria oxygen consumption and hepatic nicotinamide adenine dinucleotide (NAD + ), acylcarnitine, and ketone levels, which were attenuated in L-DRP1 KO mice, suggesting that loss of hepatic DRP1 leads to maladaptation to alcohol-induced metabolic stress. RNA-sequencing and real-time quantitative PCR analysis revealed increased gene expression of the cGAS-stimulator of interferon genes (STING)-interferon pathway in L-DRP1 KO mice regardless of alcohol feeding. Alcohol-fed L-DRP1 KO mice had increased cytosolic mtDNA and mitochondrial dysfunction leading to increased activation of cGAS-STING-interferon signaling pathways and liver injury.
CONCLUSION
Alcohol consumption decreases hepatic DRP1 resulting in increased megamitochondria and mitochondrial maladaptation that promotes AH by mitochondria-mediated inflammation and cell injury.
Topics: Mice; Humans; Animals; Hepatitis, Alcoholic; Mitochondrial Swelling; Liver Diseases, Alcoholic; Mitochondria; Ethanol; Nucleotidyltransferases; Inflammation; Interferons; Mitochondrial Dynamics
PubMed: 35698731
DOI: 10.1002/hep.32604 -
Cell Biology and Toxicology Apr 2023Mitochondrial metabolism and function are modulated by changes in matrix Ca. Small increases in the matrix Ca stimulate mitochondrial bioenergetics, whereas excessive Ca...
Mitochondrial metabolism and function are modulated by changes in matrix Ca. Small increases in the matrix Ca stimulate mitochondrial bioenergetics, whereas excessive Ca leads to cell death by causing massive matrix swelling and impairing the structural and functional integrity of mitochondria. Sustained opening of the non-selective mitochondrial permeability transition pores (PTP) is the main mechanism responsible for mitochondrial Ca overload that leads to mitochondrial dysfunction and cell death. Recent studies suggest the existence of two or more types of PTP, and adenine nucleotide translocator (ANT) and FF-ATP synthase were proposed to form the PTP independent of each other. Here, we elucidated the role of ANT in PTP opening by applying both experimental and computational approaches. We first developed and corroborated a detailed model of the ANT transport mechanism including the matrix (ANT), cytosolic (ANT), and pore (ANT) states of the transporter. Then, the ANT model was incorporated into a simple, yet effective, empirical model of mitochondrial bioenergetics to ascertain the point when Ca overload initiates PTP opening via an ANT switch-like mechanism activated by matrix Ca and is inhibited by extra-mitochondrial ADP. We found that encoding a heterogeneous Ca response of at least three types of PTPs, weakly, moderately, and strongly sensitive to Ca, enabled the model to simulate Ca release dynamics observed after large boluses were administered to a population of energized cardiac mitochondria. Thus, this study demonstrates the potential role of ANT in PTP gating and proposes a novel mechanism governing the cryptic nature of the PTP phenomenon.
Topics: Mitochondrial Membrane Transport Proteins; Adenine Nucleotides; Mitochondrial Swelling; Mitochondria; Mitochondrial Permeability Transition Pore; Calcium
PubMed: 35606662
DOI: 10.1007/s10565-022-09724-2 -
Methods in Molecular Biology (Clifton,... 2022The loss of mitochondrial cristae integrity and mitochondrial swelling are hallmarks of multiple forms of necrotic cell death. One of the most well-studied and relevant...
The loss of mitochondrial cristae integrity and mitochondrial swelling are hallmarks of multiple forms of necrotic cell death. One of the most well-studied and relevant inducers of mitochondrial swelling is matrix calcium (Ca). Respiring mitochondria will intake available Ca into their matrix until a threshold is reached which triggers the opening of the mitochondrial permeability transition pore (MPTP). Upon opening of the pore, mitochondrial membrane potential dissipates and the mitochondria begin to swell, rendering them dysfunctional. The total amount of Ca taken up by a mitochondrion prior to the engagement of the MPTP is referred to as mitochondrial Ca retention capacity (CRC). The CRC/swelling assay is a useful tool for observing the dose-dependent event of mitochondrial dysfunction in real-time. In this technique, isolated mitochondria are treated with specific boluses of Ca until they reach CRC and undergo swelling. A fluorometer is utilized to detect an increase in transmitted light passing through the sample as the mitochondria lose cristae density, and simultaneously measures calcium uptake by way of a Ca-specific membrane impermeable fluorescent dye. Here we provide a detailed protocol describing the mitochondrial CRC/swelling assay and we discuss how varying amounts of mitochondria and Ca added to the system affect the dose-dependency of the assay. We also report how to validate the assay by using MPTP and calcium uptake inhibitors and troubleshooting common mistakes that occur with this approach.
Topics: Calcium; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Permeability
PubMed: 35771440
DOI: 10.1007/978-1-0716-2309-1_9 -
Journal of Visualized Experiments : JoVE May 2018The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca buffering,...
The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca concentration. Ca retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca leads to the permeability of the inner mitochondrial membrane, termed the opening of mitochondrial permeability transition pore (mPTP), which leads to the leakage of molecules with a molecular weight less than 1.5 kDa. Ca-triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca retention capacity and Ca-triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca are added, all steps can be completed in one day and recorded by a microplate reader. Thus, these two simple and effective assays can be adopted to assess the Ca-related mitochondrial functions.
Topics: Biological Assay; Calcium; Humans; Mitochondrial Swelling; Protein Biosynthesis
PubMed: 29781984
DOI: 10.3791/56236 -
Physiological Reports Apr 2018Kidney proximal tubules (PTs) contain a high density of mitochondria, which are required to generate ATP to power solute transport. Mitochondrial dysfunction is...
Kidney proximal tubules (PTs) contain a high density of mitochondria, which are required to generate ATP to power solute transport. Mitochondrial dysfunction is implicated in the pathogenesis of numerous kidney diseases. Damaged mitochondria are thought to produce excess reactive oxygen species (ROS), which can lead to oxidative stress and activation of cell death pathways. MitoQ is a mitochondrial targeted anti-oxidant that has shown promise in preclinical models of renal diseases. However, recent studies in nonkidney cells have suggested that MitoQ might also have adverse effects. Here, using a live imaging approach, and both in vitro and ex vivo models, we show that MitoQ induces rapid swelling and depolarization of mitochondria in PT cells, but these effects were not observed with SS-31, another targeted anti-oxidant. MitoQ consists of a lipophilic cation (Tetraphenylphosphonium [TPP]) joined to an anti-oxidant component (quinone) by a 10-carbon alkyl chain, which is thought to insert into the inner mitochondrial membrane (IMM). We found that mitochondrial swelling and depolarization was also induced by dodecyltriphenylphosphomium (DTPP), which consists of TPP and the alkyl chain, but not by TPP alone. Surprisingly, MitoQ-induced mitochondrial swelling occurred in the absence of a decrease in oxygen consumption rate. We also found that DTPP directly increased the permeability of artificial liposomes with a cardiolipin content similar to that of the IMM. In summary, MitoQ causes mitochondrial swelling and depolarization in PT cells by a mechanism unrelated to anti-oxidant activity, most likely because of increased IMM permeability due to insertion of the alkyl chain.
Topics: Animals; Antioxidants; Cells, Cultured; Kidney Tubules, Proximal; Mice; Mitochondria; Mitochondrial Swelling; Opossums; Organophosphorus Compounds; Ubiquinone
PubMed: 29611340
DOI: 10.14814/phy2.13667 -
Mitochondrion Jul 2023As the cell's energy factory and metabolic hub, mitochondria are critical for ATP synthesis to maintain cellular function. Mitochondria are highly dynamic organelles... (Review)
Review
As the cell's energy factory and metabolic hub, mitochondria are critical for ATP synthesis to maintain cellular function. Mitochondria are highly dynamic organelles that continuously undergo fusion and fission to alter their size, shape, and position, with mitochondrial fusion and fission being interdependent to maintain the balance of mitochondrial morphological changes. However, in response to metabolic and functional damage, mitochondria can grow in size, resulting in a form of abnormal mitochondrial morphology known as megamitochondria. Megamitochondria are characterized by their considerably larger size, pale matrix, and marginal cristae structure and have been observed in various human diseases. In energy-intensive cells like hepatocytes or cardiomyocytes, the pathological process can lead to the growth of megamitochondria, which can further cause metabolic disorders, cell damage and aggravates the progression of the disease. Nonetheless, megamitochondria can also form in response to short-term environmental stimulation as a compensatory mechanism to support cell survival. However, extended stimulation can reverse the benefits of megamitochondria leading to adverse effects. In this review, we will focus on the findings of the different roles of megamitochondria, and their link to disease development to identify promising clinical therapeutic targets.
Topics: Humans; Mitochondrial Swelling; Mitochondria; Metabolic Diseases; Hepatocytes; Mitochondrial Membranes; Mitochondrial Dynamics
PubMed: 37276954
DOI: 10.1016/j.mito.2023.06.001 -
Circulation Research May 2024During myocardial ischemia/reperfusion (I/R) injury, high levels of matrix Ca and reactive oxygen species (ROS) induce the opening of the mitochondrial permeability...
BACKGROUND
During myocardial ischemia/reperfusion (I/R) injury, high levels of matrix Ca and reactive oxygen species (ROS) induce the opening of the mitochondrial permeability transition pore (mPTP), which causes mitochondrial dysfunction and ultimately necrotic death. However, the mechanisms of how these triggers individually or cooperatively open the pore have yet to be determined.
METHODS
Here, we use a combination of isolated mitochondrial assays and in vivo I/R surgery in mice. We challenged isolated liver and heart mitochondria with Ca, ROS, and Fe to induce mitochondrial swelling. Using inhibitors of the mPTP (cyclosporine A or ADP) lipid peroxidation (ferrostatin-1, MitoQ), we determined how the triggers elicit mitochondrial damage. Additionally, we used the combination of inhibitors during I/R injury in mice to determine if dual inhibition of these pathways is additivity protective.
RESULTS
In the absence of Ca, we determined that ROS fails to trigger mPTP opening. Instead, high levels of ROS induce mitochondrial dysfunction and rupture independently of the mPTP through lipid peroxidation. As expected, Ca in the absence of ROS induces mPTP-dependent mitochondrial swelling. Subtoxic levels of ROS and Ca synergize to induce mPTP opening. Furthermore, this synergistic form of Ca- and ROS-induced mPTP opening persists in the absence of CypD (cyclophilin D), suggesting the existence of a CypD-independent mechanism for ROS sensitization of the mPTP. These ex vivo findings suggest that mitochondrial dysfunction may be achieved by multiple means during I/R injury. We determined that dual inhibition of the mPTP and lipid peroxidation is significantly more protective against I/R injury than individually targeting either pathway alone.
CONCLUSIONS
In the present study, we have investigated the relationship between Ca and ROS, and how they individually or synergistically induce mitochondrial swelling. Our findings suggest that Ca mediates mitochondrial damage through the opening of the mPTP, although ROS mediates its damaging effects through lipid peroxidation. However, subtoxic levels both Ca and ROS can induce mPTP-mediated mitochondrial damage. Targeting both of these triggers to preserve mitochondria viability unveils a highly effective therapeutic approach for mitigating I/R injury.
Topics: Animals; Lipid Peroxidation; Mitochondrial Permeability Transition Pore; Reactive Oxygen Species; Mice; Mitochondria, Heart; Male; Myocardial Reperfusion Injury; Mice, Inbred C57BL; Mitochondrial Membrane Transport Proteins; Mitochondria, Liver; Calcium; Mitochondrial Swelling
PubMed: 38618716
DOI: 10.1161/CIRCRESAHA.123.323882 -
Mitochondrion Jan 2018Mitochondria are critical players involved in both cell life and death through multiple pathways. Structural integrity, metabolism and function of mitochondria are... (Review)
Review
Mitochondria are critical players involved in both cell life and death through multiple pathways. Structural integrity, metabolism and function of mitochondria are regulated by matrix volume due to physiological changes of ion homeostasis in cellular cytoplasm and mitochondria. Ca and K presumably play a critical role in physiological and pathological swelling of mitochondria when increased uptake (influx)/decreased release (efflux) of these ions enhances osmotic pressure accompanied by high water accumulation in the matrix. Changes in the matrix volume in the physiological range have a stimulatory effect on electron transfer chain and oxidative phosphorylation to satisfy metabolic requirements of the cell. However, excessive matrix swelling associated with the sustained opening of mitochondrial permeability transition pores (PTP) and other PTP-independent mechanisms compromises mitochondrial function and integrity leading to cell death. The mechanisms of transition from reversible (physiological) to irreversible (pathological) swelling of mitochondria remain unknown. Mitochondrial swelling is involved in the pathogenesis of many human diseases such as neurodegenerative and cardiovascular diseases. Therefore, modeling analysis of the swelling process is important for understanding the mechanisms of cell dysfunction. This review attempts to describe the role of mitochondrial swelling in cell life and death and the main mechanisms involved in the maintenance of ion homeostasis and swelling. The review also summarizes and discusses different kinetic models and approaches that can be useful for the development of new models for better simulation and prediction of in vivo mitochondrial swelling.
Topics: Cardiovascular Diseases; Electron Transport; Humans; Mitochondria; Mitochondrial Membranes; Mitochondrial Swelling; Models, Biological; Neurodegenerative Diseases; Osmotic Pressure; Oxidative Phosphorylation; Permeability
PubMed: 28802667
DOI: 10.1016/j.mito.2017.08.004 -
Romanian Journal of Morphology and... 2017Osteopontin (OPN) is involved in the regulation of calcium precipitation in the brain pathology including ischemia. A 3-Nitropropionic acid (3NP) irreversibly inhibits...
Osteopontin (OPN) is involved in the regulation of calcium precipitation in the brain pathology including ischemia. A 3-Nitropropionic acid (3NP) irreversibly inhibits mitochondrial complex II in the electron transport chain, with subsequent loss of transmembrane potential and calcium entry into the mitochondria. The present study examined the 3NP-induced calcium elevation in mitochondria and OPN expression in the 3NP-lesioned striatum. Rats were subcutaneously injected 3NP (15 mg÷kg) every other day for six weeks. Histological analysis, including the Hematoxylin-Eosin, Nissl, and Alizarin Red S stainings, was performed to examine the neurotoxic effects of 3NP. The expression of OPN in the striatum of 3NP-treated rats was investigated with immunohistochemistry and immunoelectron microscopy. In the striatal lesions, extensive loss of neurons and white matter bundles was detected. OPN was mainly detected in the penumbra region of the 3NP lesion. Scattered OPN expression was colocalized in the striatal neurons. After Alizarin Red S staining, the increase of calcium deposition was detected in the striatal lesions. In the electron microscopic analysis, the localization of OPN was clearly observed in the ultrastructure of mitochondria by immunoperoxidase and immunogold-silver staining techniques. Taken together, present findings suggest that calcium-induced mitochondrial swelling is highly associated with OPN expression. Thus, striatal calcium accumulation may be derived from 3NP-induced alteration in mitochondrial calcium homeostasis and pathologically associated with the induction of OPN protein.
Topics: Animals; Brain; Disease Models, Animal; Male; Mitochondrial Swelling; Nitro Compounds; Osteopontin; Propionates; Rats; Rats, Sprague-Dawley
PubMed: 29556613
DOI: No ID Found -
Basic Research in Cardiology Sep 2023Giant mitochondria are frequently observed in different disease models within the brain, kidney, and liver. In cardiac muscle, these enlarged organelles are present... (Review)
Review
Giant mitochondria are frequently observed in different disease models within the brain, kidney, and liver. In cardiac muscle, these enlarged organelles are present across diverse physiological and pathophysiological conditions including in ageing and exercise, and clinically in alcohol-induced heart disease and various cardiomyopathies. This mitochondrial aberration is widely considered an early structural hallmark of disease leading to adverse organ function. In this thematic paper, we discuss the current state-of-knowledge on the presence, structure and functional implications of giant mitochondria in heart muscle. Despite its demonstrated reoccurrence in different heart diseases, the literature on this pathophysiological phenomenon remains relatively sparse since its initial observations in the early 60s. We review historical and contemporary investigations from cultured cardiomyocytes to human tissue samples to address the role of giant mitochondria in cardiac health and disease. Finally, we discuss their significance for the future development of novel mitochondria-targeted therapies to improve cardiac metabolism and functionality.
Topics: Humans; Myocytes, Cardiac; Mitochondrial Swelling; Mitochondria; Cardiomyopathies; Myocardium; Mitochondria, Heart
PubMed: 37775647
DOI: 10.1007/s00395-023-01011-3