Unraveling the mechanisms of cardiolipin function: The role of oxidative polymerization of unsaturated acyl chains.

Cardiolipin is a unique phospholipid of the inner mitochondrial membrane (IMM) as well as in bacteria. It performs several vital functions such as resisting osmotic rupture and stabilizing the supramolecular structure of large membrane proteins, like ATP synthases and respirasomes. The process of cardiolipin biosynthesis results in the production of immature cardiolipin. A subsequent step is required for its maturation when its acyl groups are replaced with unsaturated acyl chains, primarily linoleic acid. Linoleic acid is the major fatty acid of cardiolipin across all organs and tissues, except for the brain. Linoleic acid is not synthesized by mammalian cells. It has the unique ability to undergo oxidative polymerization at a moderately accelerated rate compared to other unsaturated fatty acids. This property can enable cardiolipin to form covalently bonded net-like structures essential for maintaining the complex geometry of the IMM and gluing the quaternary structure of large IMM protein complexes. Unlike triglycerides, phospholipids possess only two covalently linked acyl chains, which constrain their capacity to develop robust and complicated structures through oxidative polymerization of unsaturated acyl chains. Cardiolipin, on the other hand, has four fatty acids at its disposal to form covalently bonded polymer structures. Despite its significance, the oxidative polymerization of cardiolipin has been overlooked due to the negative perception surrounding biological oxidation and methodological difficulties. Here, we discuss an intriguing hypothesis that oxidative polymerization of cardiolipin is essential for the structure and function of cardiolipin in the IMM in physiological conditions. In addition, we highlight current challenges associated with the identification and characterization of oxidative polymerization of cardiolipin in vivo. Altogether, the study provides a better understanding of the structural and functional role of cardiolipin in mitochondria.

The Effects of Amyloid-ß on Metabolomic Profiles of Cardiomyocytes and Coronary Endothelial Cells.

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-ß (Aß) in the pathogenesis of cardiac dysfunction in Alzheimer's disease remain unknown. We have recently shown the effects of Aß1 - 40 and Aß1 - 42 on cell viability and mitochondrial function in cardiomyocytes and coronary artery endothelial cells. OBJECTIVE: In this study, we investigated the effects of Aß1 - 40 and Aß1 - 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 Aß1 - 40 and Aß1 - 42. In addition, we determined mitochondrial respiration and lipid peroxidation in these cells. RESULTS: We found that the metabolism of different amino acids was affected by Aß1 - 42 in each cell type, whereas the fatty acid metabolism is consistently disrupted in both types of cells. Lipid peroxidation was significantly increased, whereas mitochondrial respiration was reduced in both cell types in response to Aß1 - 42. CONCLUSION: This study revealed the disruptive effects of Aß on lipid metabolism and mitochondria function in cardiac cells.

Beta-Amyloid Instigates Dysfunction of Mitochondria in Cardiac Cells.

Alzheimer's disease (AD) includes the formation of extracellular deposits comprising aggregated ß-amyloid (Aß) fibers associated with oxidative stress, inflammation, mitochondrial abnormalities, and neuronal loss. There is an associative link between AD and cardiac diseases; however, the mechanisms underlying the potential role of AD, particularly Aß in cardiac cells, remain unknown. Here, we investigated the role of mitochondria in mediating the effects of Aß1-40 and Aß1-42 in cultured cardiomyocytes and primary coronary endothelial cells. Our results demonstrated that Aß1-40 and Aß1-42 are differently accumulated in cardiomyocytes and coronary endothelial cells. Aß1-42 had more adverse effects than Aß1-40 on cell viability and mitochondrial function in both types of cells. Mitochondrial and cellular ROS were significantly increased, whereas mitochondrial membrane potential and calcium retention capacity decreased in both types of cells in response to Aß1-42. Mitochondrial dysfunction induced by Aß was associated with apoptosis of the cells. The effects of Aß1-42 on mitochondria and cell death were more evident in coronary endothelial cells. In addition, Aß1-40 and Aß1-42 significantly increased Ca2+ -induced swelling in mitochondria isolated from the intact rat hearts. In conclusion, this study demonstrates the toxic effects of Aß on cell survival and mitochondria function in cardiac cells.

Effects of Ferroptosis on the Metabolome in Cardiac Cells: The Role of Glutaminolysis.

Ferroptosis is a novel iron-dependent regulated cell death mechanism that affects cell metabolism; however, a detailed metabolomic analysis of ferroptotic cells is not yet available. Here, we elucidated the metabolome of H9c2 cardioblasts by gas chromatography-mass spectrometry during ferroptosis induced by RSL3, a GPX4 inhibitor, in the presence of ferrostatin-1 (a ferroptosis inhibitor), XJB-5-131 (a mitochondrial-targeted ROS scavenger), or TSM-1005-44 (a newly developed cellular ROS scavenger). Results demonstrated that RSL3 decreased the levels of amino acids involved in glutathione synthesis more than two-fold. In contrast, saturated fatty acids levels were markedly increased in RSL3-challenged cells, with no effects on unsaturated fatty acids. RSL3 significantly altered the levels of mitochondrial tricarboxylic acid cycle intermediates; isocitrate and 2-oxoglutarate were found to increase, whereas succinate was significantly decreased in RSL3-challenged cells. Ferrostatin-1, XJB-5-131, and TSM-1005-44 prevented RSL3-induced cell death and conserved the metabolomic profile of the cells. Since 2-oxoglutarate is involved in the regulation of ferroptosis, particularly through glutamine metabolism, we further assessed the role of glutaminolysis in ferroptosis in H9c2 cardioblasts. Genetic silencing of GLS1, which encodes the K-type mitochondrial glutaminase (glutaminase C), protected against ferroptosis in the early stage. In conclusion, our study demonstrates that RSL3-induced ferroptosis impairs the metabolome of H9c2 cardioblasts.

Analysis of Mitochondrial Calcium Retention Capacity in Cultured Cells: Permeabilized Cells Versus Isolated Mitochondria.

In response to various pathological stimuli, such as oxidative and energy stress accompanied by high Ca2+, mitochondria undergo permeability transition (PT) leading to the opening of the non-selective PT pores (PTP) in the inner mitochondrial membrane. Opening of the pores at high conductance allows the passage of ions and solutes <1.5 kD across the membrane, that increases colloid osmotic pressure in the matrix leading to excessive mitochondrial swelling. Calcium retention capacity (CRC) reflects maximum Ca2+ overload of mitochondria that occurs just before PTP opening. Quantification of CRC is important for elucidating the effects of different pathological stimuli and the efficacy of pharmacological agents on the mitochondria. Here, we performed a comparative analysis of CRC in mitochondria isolated from H9c2 cardioblasts, and in permeabilized H9c2 cells in situ to highlight the strengths and weaknesses of the CRC technique in isolated cell mitochondria vs. permeabilized cells. The cells were permeabilized by digitonin or saponin, and the Ca2+-sensitive fluorescence probe Calcium Green-5N was used in both preparations. Results demonstrated the interference of dye-associated fluorescence signals with saponin and the adverse effects of digitonin on mitochondria at high concentrations. Analysis of the CRC in permeabilized cells revealed a higher CRC in the saponin-permeabilized cells in comparison with the digitonin-permeabilized cells. In addition, the mitochondrial CRC in saponin-permeabilized cells was higher than in isolated mitochondria. Altogether, these data demonstrate that the quantification of the mitochondrial CRC in cultured cells permeabilized by saponin has more advantages compared to the isolated mitochondria.

Elucidating the contribution of mitochondrial glutathione to ferroptosis in cardiomyocytes.

Ferroptosis is a programmed iron-dependent cell death associated with peroxidation of lipids particularly, phospholipids. Several studies suggested a possible contribution of mitochondria to ferroptosis although the mechanisms underlying mitochondria-mediated ferroptotic pathways remain elusive. Reduced glutathione (GSH) is a central player in ferroptosis that is required for glutathione peroxidase 4 to eliminate oxidized phospholipids. Mitochondria do not produce GSH, and although the transport of GSH to mitochondria is not fully understood, two carrier proteins, the dicarboxylate carrier (DIC, SLC25A10) and the oxoglutarate carrier (OGC, SLC25A11) have been suggested to participate in GSH transport. Here, we elucidated the role of DIC and OGC as well as mitochondrial bioenergetics in ferroptosis in H9c2 cardioblasts. Results showed that mitochondria are highly sensitive to ferroptotic stimuli displaying fragmentation, and lipid peroxidation shortly after the onset of ferroptotic stimulus. Inhibition of electron transport chain complexes and oxidative phosphorylation worsened RSL3-induced ferroptosis. LC-MS/MS analysis revealed a dramatic increase in the levels of pro-ferroptotic oxygenated phosphatidylethanolamine species in mitochondria in response to RSL3 (ferroptosis inducer) and cardiac ischemia-reperfusion. Inhibition of DIC and OGC aggravated ferroptosis and increased mitochondrial ROS, membrane depolarization, and GSH depletion. Dihydrolipoic acid, an essential cofactor for several mitochondrial multienzyme complexes, attenuated ferroptosis and induced direct reduction of pro-ferroptotic peroxidized phospholipids to hydroxy-phospholipids in vitro. In conclusion, we suggest that ferroptotic stimuli diminishes mitochondrial bioenergetics and stimulates GSH depletion and glutathione peroxidase 4 inactivation leading to ferroptosis.

NSrp70 is a lymphocyte-essential splicing factor that controls thymocyte development.

Alternative pre-mRNA splicing is a critical step to generate multiple transcripts, thereby dramatically enlarging the proteomic diversity. Thus, a common feature of most alternative splicing factor knockout models is lethality. However, little is known about lineage-specific alternative splicing regulators in a physiological setting. Here, we report that NSrp70 is selectively expressed in developing thymocytes, highest at the double-positive (DP) stage. Global splicing and transcriptional profiling revealed that NSrp70 regulates the cell cycle and survival of thymocytes by controlling the alternative processing of various RNA splicing factors, including the oncogenic splicing factor SRSF1. A conditional-knockout of Nsrp1 (NSrp70-cKO) using CD4Cre developed severe defects in T cell maturation to single-positive thymocytes, due to insufficient T cell receptor (TCR) signaling and uncontrolled cell growth and death. Mice displayed severe peripheral lymphopenia and could not optimally control tumor growth. This study establishes a model to address the function of lymphoid-lineage-specific alternative splicing factor NSrp70 in a thymic T cell developmental pathway.

Mitochondrial respiratory supercomplexes in mammalian cells: structural versus functional role.

Mitochondria are recognized as the main source of ATP to meet the energy demands of the cell. ATP production occurs by oxidative phosphorylation when electrons are transported through the electron transport chain (ETC) complexes and develop the proton motive force across the inner mitochondrial membrane that is used for ATP synthesis. Studies since the 1960s have been concentrated on the two models of structural organization of ETC complexes known as "solid-state" and "fluid-state" models. However, advanced new techniques such as blue-native gel electrophoresis, mass spectroscopy, and cryogenic electron microscopy for analysis of macromolecular protein complexes provided new data in favor of the solid-state model. According to this model, individual ETC complexes are assembled into macromolecular structures known as respiratory supercomplexes (SCs). A large number of studies over the last 20 years proposed the potential role of SCs to facilitate substrate channeling, maintain the integrity of individual ETC complexes, reduce electron leakage and production of reactive oxygen species, and prevent excessive and random aggregation of proteins in the inner mitochondrial membrane. However, many other studies have challenged the proposed functional role of SCs. Recently, a third model known as the "plasticity" model was proposed that partly reconciles both "solid-state" and "fluid-state" models. According to the "plasticity" model, respiratory SCs can co-exist with the individual ETC complexes. To date, the physiological role of SCs remains unknown, although several studies using tissue samples of patients or animal/cell models of human diseases revealed an associative link between functional changes and the disintegration of SC assembly. This review summarizes and discusses previous studies on the mechanisms and regulation of SC assembly under physiological and pathological conditions.

Cardiac Function is not Susceptible to Moderate Disassembly of Mitochondrial Respiratory Supercomplexes.

: Mitochondrial respiratory chain supercomplexes (RCS), particularly, the respirasome, which contains complexes I, III, and IV, have been suggested to participate in facilitating electron transport, reducing the production of reactive oxygen species (ROS), and maintaining the structural integrity of individual electron transport chain (ETC) complexes. Disassembly of the RCS has been observed in Barth syndrome, neurodegenerative and cardiovascular diseases, diabetes mellitus, and aging. However, the physiological role of RCS in high energy-demanding tissues such as the heart remains unknown. This study elucidates the relationship between RCS assembly and cardiac function. Adult male Sprague Dawley rats underwent Langendorff retrograde perfusion in the presence and absence of ethanol, isopropanol, or rotenone (an ETC complex I inhibitor). We found that ethanol had no effects on cardiac function, whereas rotenone reduced heart contractility, which was not recovered when rotenone was excluded from the perfusion medium. Blue native polyacrylamide gel electrophoresis revealed significant reductions of respirasome levels in ethanol- or rotenone-treated groups compared to the control group. In addition, rotenone significantly increased while ethanol had no effect on mitochondrial ROS production. In isolated intact mitochondria in vitro, ethanol did not affect respirasome assembly; however, acetaldehyde, a byproduct of ethanol metabolism, induced dissociation of respirasome. Isopropanol, a secondary alcohol which was used as an alternative compound, had effects similar to ethanol on heart function, respirasome levels, and ROS production. In conclusion, ethanol and isopropanol reduced respirasome levels without any noticeable effect on cardiac parameters, and cardiac function is not susceptible to moderate reductions of RCS.

OPA1 regulates respiratory supercomplexes assembly: The role of mitochondrial swelling.

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.

The Role of Adenine Nucleotide Translocase in the Assembly of Respiratory Supercomplexes in Cardiac Cells.

Individual electron transport chain complexes have been shown to assemble into the supramolecular structures known as the respiratory chain supercomplexes (RCS). Several studies reported an associative link between RCS disintegration and human diseases, although the physiological role, structural integrity, and mechanisms of RCS formation remain unknown. Our previous studies suggested that the adenine nucleotide translocase (ANT), the most abundant protein of the inner mitochondrial membrane, can be involved in RCS assembly. In this study, we sought to elucidate whether ANT knockdown (KD) affects RCS formation in H9c2 cardiomyoblasts. Results showed that genetic silencing of ANT1, the main ANT isoform in cardiac cells, stimulated proliferation of H9c2 cardiomyoblasts with no effect on cell viability. ANT1 KD reduced the ΔΨm but increased total cellular ATP levels and stimulated the production of total, but not mitochondrial, reactive oxygen species. Importantly, downregulation of ANT1 had no significant effects on the enzymatic activity of individual ETC complexes I-IV; however, RCS disintegration was stimulated in ANT1 KD cells as evidenced by reduced levels of respirasome, the main RCS. The effects of ANT1 KD to induce RCS disassembly was not associated with acetylation of the exchanger. In conclusion, our study demonstrates that ANT is involved in RCS assembly.

Elucidating the contribution of ETC complexes I and II to the respirasome formation in cardiac mitochondria.

Mitochondrial electron transport chain (ETC) plays a central role in ATP synthesis, and its dysfunction is associated with human diseases. Recent studies revealed that individual ETC complexes are assembled into supercomplexes. The main supercomplex, respirasome composed of complexes I, III, and IV has been suggested to improve electron channeling and control ROS production, maintain the structural integrity of ETC complexes and prevent protein aggregation in the inner mitochondrial membrane. However, many questions related to the structural organization of the respirasome, particularly, a possible role of complexes I and II in respirasome formation remain unclear. Here, we investigated whether genetic and pharmacological inhibition of complexes I and II affect respirasome assembly in cardioblast cells and isolated cardiac mitochondria. Pharmacological inhibition of the enzymatic activity of complexes I and II stimulated disruption of the respirasome. Likewise, knockdown of the complex I subunit NDUFA11 stimulated dissociation of respirasome and reduced the activity of complexes I, III, and IV. However, silencing of the membrane-anchored SDHC subunit of complex II had no effect on the respirasome assembly but reduced the activity of complexes II and IV. Downregulation of NDUFA11 or SDHC reduced ATP production and increased mitochondrial ROS production. Overall, these studies, for the first time, provide biochemical evidence that the complex I activity, and the NDUFA11 subunit are important for assembly and stability of the respirasome. The SDHC subunit of complex II is not involved in the respirasome however the complex may play a regulatory role in respirasome formation.

Wavelength-multiplexed digital holography for quantitative phase measurement using quantum dot film.

We propose an enhanced quantitative three-dimensional measurement system using wavelength-multiplexed digital holography. To simplify the configuration, a dual-peak quantum dot wavelength converter, combined with a blue LED, is adapted as a single low-coherence light source. Rather than a conventional dual-wavelength method, which records and reconstruct the object wave for each wavelength, the proposed system can capture the holograms of two wavelengths simultaneously with fewer acquisitions, simple setup, and low noise. To verify the system's performance, the measurements of the step height sample are presented.

Dual-wavelength digital holography with a low-coherence light source based on a quantum dot film.

This Letter proposes a dual-wavelength, low-coherence digital holography system with a single light source, which utilizes a quantum dot (QD) film as a wavelength converter. By changing the size of the QDs, the proposed method easily yields higher and more uniform illumination of any target wavelength, compared with bandpass-filtered light-emitting diodes. Fabrication parameters of the QD film for better conversion efficiency are discussed. Using this light source with the dual-wavelength reconstruction method extends the efficiency and range of nanoscale three-dimensional height measurements.

Association between ROS production, swelling and the respirasome integrity in cardiac mitochondria.

Although mitochondrial Ca2+ overload and ROS production play a critical role in mitochondria-mediated cell death, a cause-effect relationship between them remains elusive. This study elucidated the crosstalk between mitochondrial swelling, ROS production, and electron transfer chain (ETC) supercomplexes in rat heart mitochondria in response to Ca2+ and tert-butyl hydroperoxide (TBH), a lipid-soluble organic peroxide. Results showed that ROS production induced by TBH was significantly increased in the presence of Ca2+ in a dose-dependent manner. TBH markedly inhibited the state 3 respiration rate with no effect on the mitochondrial swelling. Ca2+ exerted a slight effect on mitochondrial respiration that was greatly aggravated by TBH. Analysis of supercomplexes revealed a minor difference in the presence of TBH and/or Ca2+. However, incubation of mitochondria in the presence of high Ca2+ (1 mM) or inhibitors of ETC complexes (rotenone and antimycin A) induced disintegration of the main supercomplex, respirasome. Thus, PTP-dependent swelling of mitochondria solely depends on Ca2+ but not ROS. TBH has no effect on the respirasome while Ca2+ induces disintegration of the supercomplex only at a high concentration. Intactness of individual ETC complexes I and III is important for maintenance of the structural integrity of the respirasome.

High Sensitivity of SIRT3 Deficient Hearts to Ischemia-Reperfusion Is Associated with Mitochondrial Abnormalities.

Aim: Sirtuins are NAD+-dependent deacetylases that regulate cell metabolism through protein acetylation/deacetylation, and SIRT3 is the major deacetylase among mitochondrial isoforms. Here, we elucidated the possible role of acetylation of cyclophilin D, a key regulator of the mitochondrial permeability transition pore (mPTP), in mitochondria-mediated cardiac dysfunction induced by ischemia-reperfusion (IR) in wild type (WT) and SIRT3 knockout (SIRT3-/-) mice. Materials and Methods: Isolated and Langendorff-mode perfused hearts of WT and SIRT3-/- mice were subjected to 25-min global ischemia followed by 60-min of reperfusion in the presence or absence of the mPTP inhibitor, sanglifehrin A (SfA). Results: Analysis of mitochondrial sirtuins demonstrated that SIRT3 deficiency upregulated SIRT4 with no effect on SIRT5 expression. Hearts of SIRT3-/- mice exhibited significantly less recovery of cardiac function at the end of IR compared to WT mice. Intact (non-perfused) SIRT3-/- hearts exhibited an increased rate of Ca2+-induced swelling in mitochondria as an indicator of mPTP opening. However, there was no difference in mPTP opening and cyclophilin D acetylation between WT and SIRT3-/- hearts subjected to IR injury. Ca2+-stimulated H2O2 production was significantly higher in SIRT3-/- mitochondria that was prevented by SfA. Superoxide dismutase activity was lower in SIRT3-/- heart mitochondria subjected to IR which correlated with an increase in protein carbonylation. However, mitochondrial DNA integrity was not affected in SIRT3-/- hearts after IR. Conclusion: SIRT3 deficiency exacerbates cardiac dysfunction during post-ischemic recovery, and increases mPTP opening and ROS generation without oxidative damage to mitochondrial proteins and DNA.

Corrigendum: High Sensitivity of SIRT3 Deficient Hearts to Ischemia-Reperfusion Is Associated with Mitochondrial Abnormalities.

[This corrects the article on p. 275 in vol. 8, PMID: 28559847.].

Mitochondrial permeability transition in cardiac ischemia-reperfusion: whether cyclophilin D is a viable target for cardioprotection?

Growing number of studies provide strong evidence that the mitochondrial permeability transition pore (PTP), a non-selective channel in the inner mitochondrial membrane, is involved in the pathogenesis of cardiac ischemia-reperfusion and can be targeted to attenuate reperfusion-induced damage to the myocardium. The molecular identity of the PTP remains unknown and cyclophilin D is the only protein commonly accepted as a major regulator of the PTP opening. Therefore, cyclophilin D is an attractive target for pharmacological or genetic therapies to reduce ischemia-reperfusion injury in various animal models and humans. Most animal studies demonstrated cardioprotective effects of PTP inhibition; however, a recent large clinical trial conducted by international groups demonstrated that cyclosporine A, a cyclophilin D inhibitor, failed to protect the heart in patients with myocardial infarction. These studies, among others, raise the question of whether cyclophilin D, which plays an important physiological role in the regulation of cell metabolism and mitochondrial bioenergetics, is a viable target for cardioprotection. This review discusses previous studies to provide comprehensive information on the physiological role of cyclophilin D as well as PTP opening in the cell that can be taken into consideration for the development of new PTP inhibitors.