Membrane remodeling by FAM92A1 during brain development regulates neuronal morphology, synaptic function, and cognition.

The Bin/Amphiphysin/Rvs (BAR) domain protein FAM92A1 is a multifunctional protein engaged in regulating mitochondrial ultrastructure and ciliogenesis, but its physiological role in the brain remains unclear. Here, we show that FAM92A1 is expressed in neurons starting from embryonic development. FAM92A1 knockout in mice results in altered brain morphology and age-associated cognitive deficits, potentially due to neuronal degeneration and disrupted synaptic plasticity. Specifically, FAM92A1 deficiency impairs diverse neuronal membrane morphology, including the mitochondrial inner membrane, myelin sheath, and synapses, indicating its roles in membrane remodeling and maintenance. By determining the crystal structure of the FAM92A1 BAR domain, combined with atomistic molecular dynamics simulations, we uncover that FAM92A1 interacts with phosphoinositide- and cardiolipin-containing membranes to induce lipid-clustering and membrane curvature. Altogether, these findings reveal the physiological role of FAM92A1 in the brain, highlighting its impact on synaptic plasticity and neural function through the regulation of membrane remodeling and endocytic processes.

Neddylation inhibition prevents acetaminophen-induced liver damage by enhancing the anabolic cardiolipin pathway.

Drug-induced liver injury (DILI) is a significant cause of acute liver failure (ALF) and liver transplantation in the Western world. Acetaminophen (APAP) overdose is a main contributor of DILI, leading to hepatocyte cell death through necrosis. Here, we identified that neddylation, an essential post-translational modification involved in the mitochondria function, was upregulated in liver biopsies from patients with APAP-induced liver injury (AILI) and in mice treated with an APAP overdose. MLN4924, an inhibitor of the neuronal precursor cell-expressed developmentally downregulated protein 8 (NEDD8)-activating enzyme (NAE-1), ameliorated necrosis and boosted liver regeneration in AILI. To understand how neddylation interferes in AILI, whole-body biotinylated NEDD8 (bioNEDD8) and ubiquitin (bioUB) transgenic mice were investigated under APAP overdose with and without MLN4924. The cytidine diphosphate diacylglycerol (CDP-DAG) synthase TAM41, responsible for producing cardiolipin essential for mitochondrial activity, was found modulated under AILI and restored its levels by inhibiting neddylation. Understanding this ubiquitin-like crosstalk in AILI is essential for developing promising targeted inhibitors for DILI treatment.

Functional diversity among cardiolipin binding sites on the mitochondrial ADP/ATP carrier.

Lipid-protein interactions play a multitude of essential roles in membrane homeostasis. Mitochondrial membranes have a unique lipid-protein environment that ensures bioenergetic efficiency. Cardiolipin (CL), the signature mitochondrial lipid, plays multiple roles in promoting oxidative phosphorylation (OXPHOS). In the inner mitochondrial membrane, the ADP/ATP carrier (AAC in yeast; adenine nucleotide translocator, ANT in mammals) exchanges ADP and ATP, enabling OXPHOS. AAC/ANT contains three tightly bound CLs, and these interactions are evolutionarily conserved. Here, we investigated the role of these buried CLs in AAC/ANT using a combination of biochemical approaches, native mass spectrometry, and molecular dynamics simulations. We introduced negatively charged mutations into each CL-binding site of yeast Aac2 and established experimentally that the mutations disrupted the CL interactions. While all mutations destabilized Aac2 tertiary structure, transport activity was impaired in a binding site-specific manner. Additionally, we determined that a disease-associated missense mutation in one CL-binding site in human ANT1 compromised its structure and transport activity, resulting in OXPHOS defects. Our findings highlight the conserved significance of CL in AAC/ANT structure and function, directly tied to specific lipid-protein interactions.

In Vitro Hypoxia/Reoxygenation Induces Mitochondrial Cardiolipin Remodeling in Human Kidney Cells.

Renal ischemia/reperfusion is a serious condition that not only causes acute kidney injury, a severe clinical syndrome with high mortality, but is also an inevitable part of kidney transplantation or other kidney surgeries. Alterations of oxygen levels during ischemia/reperfusion, namely hypoxia/reoxygenation, disrupt mitochondrial metabolism and induce structural changes that lead to cell death. A signature mitochondrial phospholipid, cardiolipin, with many vital roles in mitochondrial homeostasis, is one of the key players in hypoxia/reoxygenation-induced mitochondrial damage. In this study, we analyze the effect of hypoxia/reoxygenation on human renal proximal tubule epithelial cell (RPTEC) cardiolipins, as well as their metabolism and mitochondrial functions. RPTEC cells were placed in a hypoxic chamber with a 2% oxygen atmosphere for 24 h to induce hypoxia; then, they were replaced back into regular growth conditions for 24 h of reoxygenation. Surprisingly, after 24 h, hypoxia cardiolipin levels substantially increased and remained higher than control levels after 24 h of reoxygenation. This was explained by significantly elevated levels of cardiolipin synthase and lysocardiolipin acyltransferase 1 (LCLAT1) gene expression and protein levels. Meanwhile, hypoxia/reoxygenation decreased ADP-dependent mitochondrial respiration rates and oxidative phosphorylation capacity and increased reactive oxygen species generation. Our findings suggest that hypoxia/reoxygenation induces cardiolipin remodeling in response to reduced mitochondrial oxidative phosphorylation in a way that protects mitochondrial function.

SS-31 treatment ameliorates cardiac mitochondrial morphology and defective mitophagy in a murine model of Barth syndrome.

Barth syndrome (BTHS) is a lethal rare genetic disorder, which results in cardiac dysfunction, severe skeletal muscle weakness, immune issues and growth delay. Mutations in the TAFAZZIN gene, which is responsible for the remodeling of the phospholipid cardiolipin (CL), lead to abnormalities in mitochondrial membrane, including alteration of mature CL acyl composition and the presence of monolysocardiolipin (MLCL). The dramatic increase in the MLCL/CL ratio is the hallmark of patients with BTHS, which is associated with mitochondrial bioenergetics dysfunction and altered membrane ultrastructure. There are currently no specific therapies for BTHS. Here, we showed that cardiac mitochondria isolated from TAFAZZIN knockdown (TazKD) mice presented abnormal ultrastructural membrane morphology, accumulation of vacuoles, pro-fission conditions and defective mitophagy. Interestingly, we found that in vivo treatment of TazKD mice with a CL-targeted small peptide (named SS-31) was able to restore mitochondrial morphology in tafazzin-deficient heart by affecting specific proteins involved in dynamic process and mitophagy. This agrees with our previous data showing an improvement in mitochondrial respiratory efficiency associated with increased supercomplex organization in TazKD mice under the same pharmacological treatment. Taken together our findings confirm the beneficial effect of SS-31 in the amelioration of tafazzin-deficient dysfunctional mitochondria in a BTHS animal model.

Genetic evidence for functional diversification of gram-negative intermembrane phospholipid transporters.

The outer membrane of gram-negative bacteria is a barrier to chemical and physical stress. Phospholipid transport between the inner and outer membranes has been an area of intense investigation and, in E. coli K-12, it has recently been shown to be mediated by YhdP, TamB, and YdbH, which are suggested to provide hydrophobic channels for phospholipid diffusion, with YhdP and TamB playing the major roles. However, YhdP and TamB have different phenotypes suggesting distinct functions. It remains unclear whether these functions are related to phospholipid metabolism. We investigated a synthetic cold sensitivity caused by deletion of fadR, a transcriptional regulator controlling fatty acid degradation and unsaturated fatty acid production, and yhdP, but not by ΔtamB ΔfadR or ΔydbH ΔfadR. Deletion of tamB recuses the ΔyhdP ΔfadR cold sensitivity further demonstrating the phenotype is related to functional diversification between these genes. The ΔyhdP ΔfadR strain shows a greater increase in cardiolipin upon transfer to the non-permissive temperature and genetically lowering cardiolipin levels can suppress cold sensitivity. These data also reveal a qualitative difference between cardiolipin synthases in E. coli, as deletion of clsA and clsC suppresses cold sensitivity but deletion of clsB does not. Moreover, increased fatty acid saturation is necessary for cold sensitivity and lowering this level genetically or through supplementation of oleic acid suppresses the cold sensitivity of the ΔyhdP ΔfadR strain. Together, our data clearly demonstrate that the diversification of function between YhdP and TamB is related to phospholipid metabolism. Although indirect regulatory effects are possible, we favor the parsimonious hypothesis that YhdP and TamB have differential phospholipid-substrate transport preferences. Thus, our data provide a potential mechanism for independent control of the phospholipid composition of the inner and outer membranes in response to changing conditions based on regulation of abundance or activity of YhdP and TamB.

Simultaneous Native Mass Spectrometry Analysis of Single and Double Mutants To Probe Lipid Binding to Membrane Proteins.

Lipids are critical modulators of membrane protein structure and function. However, it is challenging to investigate the thermodynamics of protein-lipid interactions because lipids can simultaneously bind membrane proteins at different sites with different specificities. Here, we developed a native mass spectrometry (MS) approach using single and double mutants to measure the relative energetic contributions of specific residues on Aquaporin Z (AqpZ) toward cardiolipin (CL) binding. We first mutated potential lipid-binding residues on AqpZ, and mixed mutant and wild-type proteins together with CL. By using native MS to simultaneously resolve lipid binding to the mutant and wild-type proteins in a single spectrum, we directly determined the relative affinities of CL binding, thereby revealing the relative Gibbs free energy change for lipid binding caused by the mutation. Comparing different mutants revealed that W14 contributes to the tightest CL binding site, with R224 contributing to a lower affinity site. Using double mutant cycling, we investigated the synergy between W14 and R224 sites on CL binding. Overall, this novel native MS approach provides unique insights into the binding of lipids to specific sites on membrane proteins.

Cardiolipin in myocardial ischaemia-reperfusion injury: From molecular mechanisms to clinical strategies.

Myocardial reperfusion injury occurs when blood flow is restored after ischemia, an essential process to salvage ischemic tissue. However, this phenomenon is intricate, characterized by various harmful effects. Tissue damage in ischemia-reperfusion injury arises from various factors, including the production of reactive oxygen species, the sequestration of proinflammatory immune cells in ischemic tissues, the induction of endoplasmic reticulum stress, and the occurrence of postischemic capillary no-reflow. Secretory phospholipase A2 (sPLA2) plays a crucial role in the eicosanoid pathway by releasing free arachidonic acid from membrane phospholipids' sn-2 position. This liberated arachidonic acid serves as a substrate for various eicosanoid biosynthetic enzymes, including cyclooxygenases, lipoxygenases, and cytochromes P450, ultimately resulting in inflammation and an elevated risk of reperfusion injury. Therefore, the activation of sPLA2 directly correlates with the heightened and accelerated damage observed in myocardial ischemia-reperfusion injury (MIRI). Presently, clinical trials are in progress for medications aimed at sPLA2, presenting promising avenues for intervention. Cardiolipin (CL) plays a crucial role in maintaining mitochondrial function, and its alteration is closely linked to mitochondrial dysfunction observed in MIRI. This paper provides a critical analysis of CL modifications concerning mitochondrial dysfunction in MIRI, along with its associated molecular mechanisms. Additionally, it delves into various pharmacological approaches to prevent or alleviate MIRI, whether by directly targeting mitochondrial CL or through indirect means.

Elucidating Daptomycin's Antibacterial Efficacy: Insights into the Tripartite Complex with Lipid II and Phospholipids in Bacterial Septum Membrane.

This study elucidated the mechanism of formation of a tripartite complex containing daptomycin (Dap), lipid II, and phospholipid phosphatidylglycerol in the bacterial septum membrane, which was previously reported as the cause of the antibacterial action of Dap against gram-positive bacteria via molecular dynamics and enhanced sampling methods. Others have suggested that this transient complex ushers in the inhibition of cell wall synthesis by obstructing the downstream polymerization and cross-linking processes involving lipid II, which is absent in the presence of cardiolipin lipid in the membrane. In this work, we observed that the complex was stabilized by Ca2+-mediated electrostatic interactions between Dap and lipid head groups, hydrophobic interaction, hydrogen bonds, and salt bridges between the lipopeptide and lipids and was associated with Dap concentration-dependent membrane depolarization, thinning of the bilayer, and increased lipid tail disorder. Residues Orn6 and Kyn13, along with the DXDG motif, made simultaneous contact with constituent lipids, hence playing a crucial role in the formation of the complex. Incorporating cardiolipin into the membrane model led to its competitively displacing lipid II away from the Dap, reducing the lifetime of the complex and the nonexistence of lipid tail disorder and membrane depolarization. No evidence of water permeation inside the membrane hydrophobic interior was noted in all of the systems studied. Additionally, it was shown that using hydrophobic contacts between Dap and lipids as collective variables for enhanced sampling gave rise to a free energy barrier for the translocation of the lipopeptide. A better understanding of Dap's antibacterial mechanism, as studied through this work, will help develop lipopeptide-based antibiotics for rising Dap-resistant bacteria.

Cardiolipin oxidized by ROS from complex II acts as a target of gasdermin D to drive mitochondrial pore and heart dysfunction in endotoxemia.

Cardiac dysfunction, an early complication of endotoxemia, is the major cause of death in intensive care units. No specific therapy is available at present for this cardiac dysfunction. Here, we show that the N-terminal gasdermin D (GSDMD-N) initiates mitochondrial apoptotic pore and cardiac dysfunction by directly interacting with cardiolipin oxidized by complex II-generated reactive oxygen species (ROS) during endotoxemia. Caspase-4/11 initiates GSDMD-N pores that are subsequently amplified by the upregulation and activation of NLRP3 inflammation through further generation of ROS. GSDMD-N pores form prior to BAX and VDAC1 apoptotic pores and further incorporate into BAX and VDAC1 oligomers within mitochondria membranes to exacerbate the apoptotic process. Our findings identify oxidized cardiolipin as the definitive target of GSDMD-N in mitochondria of cardiomyocytes during endotoxin-induced myocardial dysfunction (EIMD), and modulation of cardiolipin oxidation could be a therapeutic target early in the disease process to prevent EIMD.

Elucidation of molecular mechanisms by which amyloid ß1-42 fibrils exert cell toxicity.

Abrupt aggregation of amyloid ß1-42 (Aß1-42) peptide in the frontal lobe is the expected underlying cause of Alzheimer's disease (AD). ß-Sheet-rich oligomers and fibrils formed by Aß1-42 exert high cell toxicity. A growing body of evidence indicates that lipids can uniquely alter the secondary structure and toxicity of Aß1-42 aggregates. At the same time, underlying molecular mechanisms that determine this difference in toxicity of amyloid aggregates remain unclear. Using a set of molecular and biophysical assays to determine the molecular mechanism by which Aß1-42 aggregates formed in the presence of cholesterol, cardiolipin, and phosphatidylcholine exert cell toxicity. Our findings demonstrate that rat neuronal cells exposed to Aß1-42 fibrils formed in the presence of lipids with different chemical structure exert drastically different magnitude and dynamic of unfolded protein response (UPR) in the endoplasmic reticulum (ER) and mitochondria (MT). We found that the opposite dynamics of UPR in MT and ER in the cells exposed to Aß1-42: cardiolipin fibrils and Aß1-42 aggregates formed in a lipid-free environment. We also found that Aß1-42: phosphatidylcholine fibrils upregulated ER UPR simultaneously downregulating the UPR response of MT, whereas Aß1-42: cholesterol fibrils suppressed the UPR response of ER and upregulated UPR response of MT. We also observed progressively increasing ROS production that damages mitochondrial membranes and other cell organelles, ultimately leading to cell death.

The Role of Cardiolipin in Mitochondrial Function and Neurodegenerative Diseases.

Cardiolipin (CL) is a mitochondria-exclusive phospholipid synthesized in the inner mitochondrial membrane. CL plays a key role in mitochondrial membranes, impacting a plethora of functions this organelle performs. Consequently, it is conceivable that abnormalities in the CL content, composition, and level of oxidation may negatively impact mitochondrial function and dynamics, with important implications in a variety of diseases. This review concentrates on papers published in recent years, combined with basic and underexplored research in CL. We capture new findings on its biological functions in the mitochondria, as well as its association with neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease. Lastly, we explore the potential applications of CL as a biomarker and pharmacological target to mitigate mitochondrial dysfunction.

SIRT3 regulates cardiolipin biosynthesis in pressure overload-induced cardiac remodeling by PPARγ-mediated mechanism.

Cardiac remodeling is the primary pathological feature of chronic heart failure (HF). Exploring the characteristics of cardiac remodeling in the very early stages of HF and identifying targets for intervention are essential for discovering novel mechanisms and therapeutic strategies. Silent mating type information regulation 2 homolog 3 (SIRT3), as a major mitochondrial nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, is required for mitochondrial metabolism. However, whether SIRT3 plays a role in cardiac remodeling by regulating the biosynthesis of mitochondrial cardiolipin (CL) is unknown. In this study, we induced pressure overload in wild-type (WT) and SIRT3 knockout (SIRT3-/-) mice via transverse aortic constriction (TAC). Compared with WT mouse hearts, the hearts of SIRT3-/- mice exhibited more-pronounced cardiac remodeling and fibrosis, greater reactive oxygen species (ROS) production, decreased mitochondrial-membrane potential (ΔΨm), and abnormal mitochondrial morphology after TAC. Furthermore, SIRT3 deletion aggravated TAC-induced decrease in total CL content, which might be associated with the downregulation of the CL synthesis related enzymes cardiolipin synthase 1 (CRLS1) and phospholipid-lysophospholipid transacylase (TAFAZZIN). In our in vitro experiments, SIRT3 overexpression prevented angiotensin II (AngII)- induced aberrant mitochondrial function, CL biosynthesis disorder, and peroxisome proliferator-activated receptor gamma (PPARγ) downregulation in cardiomyocytes; meanwhile, SIRT3 knockdown exacerbated these effects. Moreover, the addition of GW9662, a PPARγ antagonist, partially counteracted the beneficial effects of SIRT3 overexpression. In conclusion, SIRT3 regulated PPARγ-mediated CL biosynthesis, maintained the structure and function of mitochondria, and thereby protected the myocardium against cardiac remodeling.

The role of cardiolipin and cytochrome c in mitochondrial metabolism of cancer cells determined by Raman imaging: in vitro study on the brain glioblastoma U-87 MG cell line.

In this paper, we present Raman imaging as a non-invasive approach for studying changes in mitochondrial metabolism caused by cardiolipin-cytochrome c interactions. We investigated the effect of mitochondrial dysregulation on cardiolipin (CL) and cytochrome c (Cyt c) interactions for a brain cancer cell line (U-87 MG). Mitochondrial metabolism was monitored by checking the intensities of the Raman bands at 750 cm-1, 1126 cm-1, 1310 cm-1, 1337 cm-1, 1444 cm-1 and 1584 cm-1. The presented results indicate that under pathological conditions, the content and redox status of Cyt c in mitochondria can be used as a Raman marker to characterize changes in cellular metabolism. This work provides evidence that cardiolipin-cytochrome c interactions are crucial for mitochondrial energy homeostasis by controlling the redox status of Cyt c in the electron transport chain, switching from disabling Cyt c reduction and enabling peroxidase activity. This paper provides experimental support for the hypothesis of how cardiolipin-cytochrome c interactions regulate electron transfer in the respiratory chain, apoptosis and mROS production in mitochondria.

Age-dependent loss of Crls1 causes myopathy and skeletal muscle regeneration failure.

Skeletal muscle aging results in the gradual suppression of myogenesis, leading to muscle mass loss. However, the specific role of cardiolipin in myogenesis has not been determined. This study investigated the crucial role of mitochondrial cardiolipin and cardiolipin synthase 1 (Crls1) in age-related muscle deterioration and myogenesis. Our findings demonstrated that cardiolipin and Crls1 are downregulated in aged skeletal muscle. Moreover, the knockdown of Crls1 in myoblasts reduced mitochondrial mass, activity, and OXPHOS complex IV expression and disrupted the structure of the mitochondrial cristae. AAV9-shCrls1-mediated downregulation of Crls1 impaired muscle regeneration in a mouse model of cardiotoxin (CTX)-induced muscle damage, whereas AAV9-mCrls1-mediated Crls1 overexpression improved regeneration. Overall, our results highlight that the age-dependent decrease in CRLS1 expression contributes to muscle loss by diminishing mitochondrial quality in skeletal muscle myoblasts. Hence, modulating CRLS1 expression is a promising therapeutic strategy for mitigating muscle deterioration associated with aging, suggesting potential avenues for developing interventions to improve overall muscle health and quality of life in elderly individuals.

The unusual substrate specificity of Escherichia coli cardiolipin synthase C does not require the product of the transcriptionally engaged ymdB gene.

Polycistronic transcription and translation of ymdB-clsC have been thought to be required for full activity of ClsC. The authentic initiation codon of the clsC gene is present within the open reading frame of the upstream located ymdB gene. ClsC translated from authentic initiation codon drives cardiolipin (CL) synthesis without transcriptionally paired YmdB. YmdB is not necessary for the substrate specificity of ClsC utilizing phosphatidylethanolamine (PE) as a co-substrate.

The phospholipids cardiolipin and phosphatidylethanolamine differentially regulate MDC biogenesis.

Cells utilize multiple mechanisms to maintain mitochondrial homeostasis. We recently characterized a pathway that remodels mitochondria in response to metabolic alterations and protein overload stress. This remodeling occurs via the formation of large membranous structures from the mitochondrial outer membrane called mitochondrial-derived compartments (MDCs), which are eventually released from mitochondria and degraded. Here, we conducted a microscopy-based screen in budding yeast to identify factors that regulate MDC formation. We found that two phospholipids, cardiolipin (CL) and phosphatidylethanolamine (PE), differentially regulate MDC biogenesis. CL depletion impairs MDC biogenesis, whereas blocking mitochondrial PE production leads to constitutive MDC formation. Additionally, in response to metabolic MDC activators, cellular and mitochondrial PE declines, and overexpressing mitochondrial PE synthesis enzymes suppress MDC biogenesis. Altogether, our data indicate a requirement for CL in MDC biogenesis and suggest that PE depletion may stimulate MDC formation downstream of MDC-inducing metabolic stress.

Decreased pyruvate dehydrogenase activity in Tafazzin-deficient cells is caused by dysregulation of pyruvate dehydrogenase phosphatase 1 (PDP1).

Cardiolipin (CL), the signature lipid of the mitochondrial inner membrane, is critical for maintaining optimal mitochondrial function and bioenergetics. Disruption of CL metabolism, caused by mutations in the CL remodeling enzyme TAFAZZIN, results in the life-threatening disorder Barth syndrome (BTHS). While the clinical manifestations of BTHS, such as dilated cardiomyopathy and skeletal myopathy, point to defects in mitochondrial bioenergetics, the disorder is also characterized by broad metabolic dysregulation, including abnormal levels of metabolites associated with the tricarboxylic acid (TCA) cycle. Recent studies have identified the inhibition of pyruvate dehydrogenase (PDH), the gatekeeper enzyme for TCA cycle carbon influx, as a key deficiency in various BTHS model systems. However, the molecular mechanisms linking aberrant CL remodeling, particularly the primary, direct consequence of reduced tetralinoleoyl-CL (TLCL) levels, to PDH activity deficiency are not yet understood. In the current study, we found that remodeled TLCL promotes PDH function by directly binding to and enhancing the activity of PDH phosphatase 1 (PDP1). This is supported by our findings that TLCL uniquely activates PDH in a dose-dependent manner, TLCL binds to PDP1 in vitro, TLCL-mediated PDH activation is attenuated in the presence of phosphatase inhibitor, and PDP1 activity is decreased in Tafazzin-knockout (TAZ-KO) C2C12 myoblasts. Additionally, we observed decreased mitochondrial calcium levels in TAZ-KO cells and treating TAZ-KO cells with calcium lactate (CaLac) increases mitochondrial calcium and restores PDH activity and mitochondrial oxygen consumption rate. Based on our findings, we conclude that reduced mitochondrial calcium levels and decreased binding of PDP1 to TLCL contribute to decreased PDP1 activity in TAZ-KO cells.

Identification of MIMAS, a multifunctional mega-assembly integrating metabolic and respiratory biogenesis factors of mitochondria.

The mitochondrial inner membrane plays central roles in bioenergetics and metabolism and contains several established membrane protein complexes. Here, we report the identification of a mega-complex of the inner membrane, termed mitochondrial multifunctional assembly (MIMAS). Its large size of 3 MDa explains why MIMAS has escaped detection in the analysis of mitochondria so far. MIMAS combines proteins of diverse functions from respiratory chain assembly to metabolite transport, dehydrogenases, and lipid biosynthesis but not the large established supercomplexes of the respiratory chain, ATP synthase, or prohibitin scaffold. MIMAS integrity depends on the non-bilayer phospholipid phosphatidylethanolamine, in contrast to respiratory supercomplexes whose stability depends on cardiolipin. Our findings suggest that MIMAS forms a protein-lipid mega-assembly in the mitochondrial inner membrane that integrates respiratory biogenesis and metabolic processes in a multifunctional platform.

Kaempferol Improves Cardiolipin and ATP in Hepatic Cells: A Cellular Model Perspective in the Context of Metabolic Dysfunction-Associated Steatotic Liver Disease.

Targeting mitochondrial function is a promising approach to prevent metabolic dysfunction-associated steatotic liver disease (MASLD). Cardiolipin (CL) is a unique lipid comprising four fatty acyl chains localized in the mitochondrial inner membrane. CL is a crucial phospholipid in mitochondrial function, and MASLD exhibits CL-related anomalies. Kaempferol (KMP), a natural flavonoid, has hepatoprotective and mitochondrial function-improving effects; however, its influence on CL metabolism in fatty liver conditions is unknown. In this study, we investigated the effects of KMP on mitochondrial function, focusing on CL metabolism in a fatty liver cell model (linoleic-acid-loaded C3A cell). KMP promoted mitochondrial respiratory functions such as ATP production, basal respiration, and proton leak. KMP also increased the gene expression levels of CPT1A and PPARGC1A, which are involved in mitochondrial ß-oxidation. Comprehensive quantification of CL species and related molecules via liquid chromatography/mass spectrometry showed that KMP increased not only total CL content but also CL72:8, which strongly favors ATP production. Furthermore, KMP improved the monolysocardiolipin (MLCL)/CL ratio, an indicator of mitochondrial function. Our results suggest that KMP promotes energy production in a fatty liver cell model, associated with improvement in mitochondrial CL profile, and can serve as a potential nutrition factor in preventing MASLD.